Bio-Oil Production Method

ABSTRACT

The invention relates to methods for the conversion of lignocellulosic matter into fuel products. More specifically, the invention relates to methods for the generation of a bio-oil product from specific component(s) of lignocellulosic matter.

INCORPORATION BY REFERENCE

This application claims priority from U.S. provisional application No.61/101,805 filed on 1 Oct. 2008, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods for the conversion of lignocellulosicmatter into fuel products. More specifically, the invention relates tomethods for the generation of a bio-oil product from specificcomponent(s) of lignocellulosic matter.

BACKGROUND

With the continuing high price of oil and its increasing importationcosts in many countries, the production of alternative fuel products(“biofuels”) is becoming increasingly important. A significant amount ofresearch in the field has focussed on the conversion of lignocellulosicmatter into fuel products such as ethanol to provide an alternative andrenewable feedstock to the depleting sources of hydrocarbon-based rawmaterials.

Lignocellulosic matter consists of carbohydrate polymers (celluloses andhemicelluloses) and the phenolic polymer lignin. Existing technologiesfor the conversion of lignocellulosic matter into fuel productsgenerally utilize a series of steps involving fractionation of thebiomass followed by saccharification and fermentation. Thesaccharification and fermentation steps are often complex and addsignificantly to the cost of the process. Further, the hydrolysis ofcellulose and hemicellulose into simple sugars suitable for fermentationis significantly hindered by the presence of tightly bound lignin.Existing technologies expend significant energy in decreasing the lignincontent of sugar-containing fractions in order to increase accessibilityby hydrolytic enzymes.

Lignin makes up a significant proportion of lignocellulosic matter andoffers another utilizable resource in addition to the cellulosic andhemicellulosic components. However, a large proportion of biomassconversion methods fail to effectively utilize the lignin componentwhich instead goes to waste. Additionally, many of the existingprocesses yield only ethanol. While ethanol is usable as a fuel, theenergy content on a volume basis is about 30% less than currently usedfossil fuels, and is not practical in current diesel engines. Ethanolalso attracts water, which makes storage and handling difficult.

A need exists for improved methods of converting lignocellulosic matterinto energy-containing products such as biofuels. A need also exists forbiofuel production methods that better exploit the energy-producingpotential of lignin.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a method for solvatinglignocellulosic biomass, the method comprising the steps of

-   -   (a) fractionating hemicellulose from the biomass with a solvent,    -   (b) removing fractionated hemicellulose from biomass remaining        after step (a); and    -   (c) solvating lignin and cellulose from the remaining biomass        with a solvent.

In a second aspect, there is provided a method for producing a bio-oilproduct from lignocellulosic biomass, the method comprising the stepsof:

-   -   (a) fractionating hemicellulose from the biomass with a solvent,    -   (b) removing fractionated hemicellulose from biomass remaining        after step (a); and    -   (c) solvating lignin and cellulose from the biomass remaining        after step (a) with a solvent,    -   wherein the solvating in step (c) produces the bio-oil product.

In a third aspect, there is provided a method for producing a bio-oilproduct from lignocellulosic biomass, the method comprising the stepsof:

-   -   (a) fractionating hemicellulose from the biomass with a solvent,    -   (b) removing fractionated hemicellulose from biomass remaining        after step (a);    -   (c) fractionating either of:        -   (i) lignin        -   (ii) cellulose    -   from the biomass remaining after step (a); and    -   (d) solvating either or both of the lignin and cellulose of step        (c),    -   wherein the solvating in step (d) produces the bio-oil product.

In one embodiment of the third aspect, the fractionating in step (c) isperformed using an alcohol, an aqueous alcohol, or water. The alcohol,aqueous alcohol, or water may be used to fractionate the lignin orcellulose under supercritical conditions.

In one embodiment of the first, second or third aspect, fractionating ofhemicellulose in step (a) is performed using sub-critical water.

In another embodiment of the first, second or third aspect,fractionating of hemicellulose using sub-critical water is performed ata temperature of between about 100° C. and about 300° C.

In an additional embodiment of the first, second or third aspect,fractionating of hemicellulose using sub-critical water is performed ata pressure of between about 2 MPa (20 bar) and about 4 MPa (40 bar).

In a further embodiment of the first, second or third aspect,fractionating of hemicellulose using sub-critical water is performed atabout 190° C. and about 3 MPa (30 bar).

In one embodiment of the first, second or third aspect, the fractionatedhemicellulose component of step (b) is subjected to saccharification toproduce a fermentable saccharide. The saccharide may be fermented toproduce an alcohol selected from the group consisting of ethanol,butanol, xylitol, mannitol, and arabinol.

In a fourth aspect, there is provided a method for producing a bio-oilproduct, the method comprising the step of solvating a materialcomprising either or both of:

-   -   (i) lignin;    -   (ii) cellulose,    -   using a solvent, wherein said solvating produces the bio-oil        product.

In one embodiment of the first, second, third or fourth aspect, thesolvating is performed using a solvent that is an alkylating agent. Thealkylating agent may be selected from the group consisting of analkylhalide, an alkylsulfate, an olefin, and an alkylphosphate. Thealkylating agent may be an alcohol. The alcohol may be a C1 to C6alcohol. The C1 to C6 alcohol may be ethanol, methanol, or butanol.

The solvent may be aqueous. The aqueous solvent may comprise at leastone percent water based on total weight of solvent. The aqueous solventmay comprise at least 80 percent water based on total weight of solvent.The aqueous solvent may comprise at least 90 percent water based ontotal weight of solvent.

In one embodiment of the first, second, third or fourth aspect, thesolvating is performed at a temperature of between about 230° C. andabout 360° C.

In another embodiment of the first, second, third or fourth aspect, thesolvating is performed at a pressure of between about 14 MPa (140 bar)and about 24 MPa (240 bar).

In one embodiment of the first, second, third or fourth aspect, thesolvating is performed at a temperature of between about 230° C. andabout 360° C., and at a pressure of between about 14 MPa (140 bar) andabout 24 MPa (240 bar).

In another embodiment of the first, second, third or fourth aspect, thesolvating is performed at a temperature of about 320° C. and a pressureof about 18 MPa (180 bar).

In one embodiment of the second, third or fourth aspect, the step ofsolvating converts substantially all of the lignin into the bio-oilproduct.

In one embodiment of the second, third or fourth aspect, the step ofsolvating converts substantially all of the cellulose into the bio-oilproduct.

In one embodiment of the second, third or fourth aspect, the step ofsolvating converts substantially all of the cellulose and substantiallyall of the lignin into the bio-oil product.

In a fifth aspect, there is provided a bio-oil product obtainable by themethod of the first, second, third or fourth aspect.

In a sixth aspect, there is provided a bio-oil product obtained by themethod of the first, second, third or fourth aspect.

The bio-oil product of any of the previous aspects may be used as abiofuel, or a biofuel additive.

In a seventh aspect, there is provided a method for producing a bio-oilfrom lignocellulosic matter, the method comprising the steps of:

-   -   (a) solvating hemicellulose from the lignocellulosic matter        using a solvent,    -   (b) removing solvated hemicellulose from solid matter remaining        after step (a); and    -   (c) solvating lignin and cellulose from the solid matter        remaining after step (a) using a solvent,        wherein step (c) of solvating lignin and cellulose produces the        bio-oil.

In one embodiment of the seventh aspect, the lignocellulosic mattercomprises 10%-35% hemicellulose, 15%-45% cellulose and 2%-35% lignin.

In one embodiment of the seventh aspect, the lignocellulosic mattercomprises 20%-35% hemicellulose, 20%-45% cellulose and 20%-35% lignin.

In another embodiment of the seventh aspect, the solvent of step (c) isan aqueous alcohol comprising no more than ten carbon atoms.

In one embodiment of the seventh aspect, the aqueous alcohol is ethanolor methanol.

In an additional embodiment of the seventh aspect, the aqueous alcoholcomprises 1%-30% alcohol by weight.

In another embodiment of the seventh aspect, the aqueous alcoholcomprises 5%-30% alcohol by weight.

In one embodiment of the seventh aspect, the aqueous alcohol comprisesabout 25% alcohol by weight.

In another embodiment of the seventh aspect, the aqueous alcoholcomprises about 20% alcohol by weight.

In one embodiment of the seventh aspect, step (c) is performed at areaction temperature of between 250° C. and 400° C.

In another embodiment of the seventh aspect, step (c) is performed at areaction temperature of between 280° C. and 350° C.

In one embodiment of the seventh aspect, step (c) is performed at atemperature of about 320° C.

In one embodiment of the seventh aspect, step (c) is performed at areaction pressure of between 12 MPa and 24 MPa.

In another embodiment of the seventh aspect, step (c) is performed at areaction pressure of about 20 MPa.

In one embodiment of the seventh aspect, the lignin and cellulose ofstep (c) is in the form of a slurry.

In one embodiment of the seventh aspect, the slurry comprises between 2%and 45% solid matter by weight.

In one embodiment of the seventh aspect, the slurry comprises between 2%and 30% solid matter by weight.

In a further embodiment of the seventh aspect, the slurry comprisesabout 5% solid matter by weight.

In one embodiment of the seventh aspect, step (c) is performed forbetween 2 minutes and 60 minutes.

In one embodiment of the seventh aspect, step (c) is performed forbetween 2 minutes and 40 minutes.

In another embodiment of the seventh aspect, step (c) is performed forbetween 5 minutes and 30 minutes.

In one embodiment of the seventh aspect, the solvating of hemicellulosein step (a) is performed at a reaction temperature of between 100° C.and 250° C., and a reaction pressure of between 0.2 MPa and 5 MPa.

In a further embodiment of the seventh aspect, the solvent of step (a)is an aqueous acid and the treatment is performed at a pH of below about6.5.

In one embodiment of the seventh aspect, the solvent of step (a) is anaqueous base and the treatment is performed at a pH of above about 7.5.

In one embodiment of the seventh aspect, the solvent of step (a) iswater.

In one embodiment of the seventh aspect, the method further comprisespre-treating the lignocellulosic matter prior to solvating hemicellulosein step (a).

In an additional embodiment of the seventh aspect, the pre-treatingcomprises producing a shiny comprising a mixture of a solvent andparticles derived from the lignocellulosic matter.

In one embodiment of the seventh aspect, the particles are between about50 microns and about 500 microns in size.

In one embodiment of the seventh aspect, the particles are between about100 microns and about 400 microns in size.

In one embodiment of the seventh aspect, the slurry comprises betweenabout 5% and about 20% lignocellulosic matter.

In a further embodiment of the seventh aspect, the lignin isfractionated from the solid matter remaining after step (a) prior toperforming step (c) of solvating to produce the bio-oil.

In one embodiment of the seventh aspect, the cellulose is fractionatedfrom the solid matter remaining after step (a) prior to performing step(c) of solvating to produce the bio-oil.

In one embodiment of the seventh aspect, the solvated hemicelluloseremoved in step (b) is subjected to saccharification to produce afermentable saccharide.

In an additional embodiment of the seventh aspect, the saccharide isfermented to produce an alcohol selected from the group consisting ofethanol, butanol, xylitol, mannitol, and arabinol.

In an eighth aspect, there is provided a method for producing a bio-oilproduct from a material comprising lignin and cellulose, the methodcomprising treating the material with a supercritical aqueous alcohol ata reaction temperature of between 180° C. and 350° C. and a reactionpressure of between 8 MPa and 26 MPa, wherein said treating solvates thelignin and cellulose producing the bio-oil product.

In one embodiment of the eighth aspect, the material is treated at areaction temperature of between 280° C. and 350° C. and a reactionpressure of between 12 MPa and 24 MPa.

In one embodiment of the eighth aspect, the aqueous alcohol comprises 1%to 30% alcohol by weight.

In one embodiment of the eighth aspect, the aqueous alcohol comprises 5%to 30% alcohol by weight.

In another embodiment of the eighth aspect, the aqueous alcohol isethanol.

In a ninth aspect, there is provided a bio-oil obtainable by the methodof the seventh or eighth aspect.

In a tenth aspect, there is provided a bio-oil obtained by the method ofthe seventh or eighth aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will now be described,by way of an example only, with reference to the accompanying drawingswherein:

FIG. 1 is a graph showing the results of a dinitrosalicyclic acid (DNS)assay conducted on hemicellulose liquor samples subjected tosaccharification using hydrolytic enzymes. Absorbance readings (I₅₄₀, inmOD) from substrate only controls and enzyme only controls weresubtracted from readings obtained from enzyme-substrate samples. Samplenumbers are shown on the horizontal axis. The vertical axis showsrelative amounts of reducing sugars present in each sample.

FIG. 2 is a graph showing the results of a gel permeation chromatography(GPC) analysis of a heavy oil fraction produced in accordance with themethods of the invention. Vertical axis: normalised intensity;horizontal axis: molecular weight; light trace: 15 minute retentiontime; dark trace: 30 minute retention time.

FIG. 3 is a graph showing the results of thermo gravimetric analysis(TGA) of a heavy oil fraction produced in accordance with the methods ofthe invention. Vertical axis: percentage of mass lost; horizontal axis:temperature (° C.); trace: represents results from heavy oil producedusing a 30 minute retention time.

FIG. 4 is a chromatogram showing the results of a gaschromatography-mass spectroscopy (GCMS) analysis of diethylether-extractable oils derived from the aqueous phase of an oil emulsionproduced in accordance with the methods of the invention. Peaks: 2.342(Ether,1-propenyl propyl), 5.600 (2-Cyclopenten-1-one, 2-methyl-), 6.949(Phenol), 8.483 (Phenol, 2-methoxy-), 9.690 (2,3-Dimethylhydroquinone),10.590 (Phenol, 4-ethyl-2-methoxy-), 10,625 (1,2-Benzenediol,4-methyl-), 11.433 (Phenol, 2-methoxy-4-propyl-), 11.731 (Vanillin),12.374 (Phenol, 2-methoxy-).

FIG. 5 is a chromatogram showing the results of a gaschromatography-mass spectroscopy (GCMS) analysis of a heavy oil fractionproduced in accordance with the methods of the invention dissolved intetrahydrofuran. Peaks: 10.585 (Phenol, 4-ethyl-2-methoxy-), 11.433(Phenol, 2-methoxy-4-propyl-), 17.067 (Oleic Acid), 17.742(2-Isopropyl-10-methylphenanthrene), 18.343(3-(3-Hydroxy-4-methoxyphenyl)-1-alanine), 18.442((−)-Nortrachelogenin), 18.686 (1-Phenanthrenecarboxylic acid,1,2,3,4,4a,9,10,10a-octahydro-1,4-a-dimethyl-7-(1-methylethyl)-, methylester, [1R-(1.alpha., 4a.beta., 10a.alpha.)]), 18.981(1-Phenanthrenecarboxylic acid,1,2,3,4,4a,9,10,10a-octahydro-1,4-a-dimethyl-7-(1-methylethyl)-,[1R-(1.alpha., 4a.beta., 10a.alpha.)]), 20.016(7-(3,4-Methylenedioxy)-tetrahydrobenzofuranone), 21.368 (Carinol).

DEFINITIONS

As used in this application, the singular form “a”, “an” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “a particle” also includes a plurality ofparticles.

As used herein, the term “comprising” means “including.” Variations ofthe word “comprising”, such as “comprise” and “comprises”, havecorrespondingly varied meanings. Thus, for example, a material“comprising” lignin and cellulose may consist exclusively of lignin andcellulose or may include other additional substances.

As used herein, the terms “lignocellulosic matter” and “lignocellulosicbiomass” are used interchangeably and have the same meaning. The termsencompass any substance comprising lignin, cellulose, and hemicellulose.

As used herein, the term “aqueous solvent” refers to a solventcontaining at least one percent water based on total weight of solvent.

As used herein, the term “aqueous ethanol” refers to an ethanol solventcontaining at least one percent water based on total weight of solvent.

As used herein, the term “saccharide” encompasses any moleculecomprising one or more monosaccharide units. Examples of saccharidesinclude, but are not limited to, cellulose, hemicellulose,polysaccharides, oligosaccharides, disaccharides and monosaccharides.“Saccharides” also include glycoconjugates, such as glycoproteins andglycolipids. All stereoisomeric and enantiomeric forms of saccharidesare encompassed by the term.

As used herein, a “supercritical” substance (e.g. a supercriticalsolvent) refers to a substance that is heated above its criticaltemperature and pressurised above its critical pressure (i.e. asubstance at a temperature and pressure above its critical point). Theterm “supercritical” also encompasses conditions of temperature and/orpressure that are a small, although not substantial, amount (e.g.approximately 5%) below the critical point of the substance in question(i.e. “sub-critical”). Accordingly, the term “supercritical” alsoencompasses oscillatory behaviour around the critical point of asubstance (i.e. movement from supercritical conditions to sub-criticalconditions and vice versa). For example, a solvent having a criticalpoint of 305 degrees Kelvin and 4.87 atmospheres may, for the purposesof the present invention, still be considered to be “supercritical” at aslightly lower temperature (e.g. between 290 degrees and 305 degreesKelvin) and/or a slightly lower pressure (e.g. between 4.63 and 4.87atmospheres).

It will be understood that use of the term “about” herein in referenceto a recited numerical value (e.g. a reaction temperature, pressure orpH) includes the recited numerical value and numerical values withinplus or minus ten percent of the recited value.

It will be understood that use of the term “between” when referring to arange of numerical values encompasses the numerical values at eachendpoint of the range. For example, a temperature range of between 10°C. and 15° C. is inclusive of the temperatures 10° C. and 15° C.

Any description of prior art documents herein, or statements hereinderived from or based on those documents, is not an admission that thedocuments or derived statements are part of the common general knowledgeof the relevant art in Australia or elsewhere.

For the purposes of description all documents referred to herein areincorporated by reference unless otherwise stated.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for generating a bio-oil product fromlignocellulosic matter without the need for enzymatic hydrolysis orfermentation. Lignocellulosic matter treated in accordance with themethods of the invention is generally subjected to a step ofhemicellulose removal followed by direct conversion of the remainingmatter (comprising predominantly lignin and cellulose) into a stablebio-oil product. The bio-oil product may be used directly, processed togenerate other products (e.g. fuels), or used as a fuel additive.Hemicellulose separated in accordance with the methods of the inventionmay be converted into products such as alcohols.

Existing technologies have demonstrated that lignocellulosic matter maybe solubilized with supercritical solvents. However, the productsgenerated often contain significant amounts of tar-like compounds andare difficult to process. The three main components of lignocellulosicmatter (i.e. lignin, cellulose and hemicellulose) are believed to havedifferent reactivities. In particular, hemicellulose is thought to beprone to excessive conversions leading to highly unstable and/or charredmaterials, whereas the other two fractions (lignin and cellulose) arebelieved to react more slowly. The high temperatures associated withsupercritical treatment are likely to induce the dissolution ofhemicellulose well before lignin and cellulose react to a significantextent. The hemicellulose-derived sugars therefore dehydrate quickly,creating double bonds and highly reactive cyclic molecules (e.g.furfural) that easily polymerise and yield tar-like compounds if notstabilised. This significantly compromises the efficiency of subsequentsteps (e.g. saccharification and fermentation) utilized in currenttechnologies to generate biofuels. The methods of the inventioncircumvent this problem by providing an initial step of hemicelluloseseparation under mild conditions thereby minimizing sugar dehydrationand the formation of tar-like molecules during the processing of thelignin and cellulose components.

The solubilisation of lignocellulosic matter using current technologiesis generally a precursor to further saccharification and fermentationsteps required for the production of biofuel. Those additional steps areoften complex and add significantly to the cost of the process. Inaddition, saccharification of solubilized cellulose and/or hemicelluloseinto sugar chains of a suitable length for fermentation is generallyhindered by the presence of tightly bound lignin. The methods of theinvention circumvent this problem by facilitating the direct conversionof lignin and cellulose into a bio-oil product without the need forsaccharification and fermentation steps.

Without being limited to a particular mechanism or mode of action, it isbelieved that treatment of matter comprising lignin and cellulose inaccordance with the methods of the invention facilitates swelling of thelignin and/or cellulose and chemical stabilization of the bio-oilproduct formed, thus minimizing polymerization into tar-like compounds.Mechanical swelling of the cellulose and/or lignin is believed to assistin “opening up” the substrate making it more accessible for hydrolysisand depolymerization. Chemical stabilization of the bio-oil product mayoccur through various interactions including alkylation and scavengingof free-radicals. For example, the alkylation of reactive groups incellulose and/or lignin is likely to prevent highly reactive speciesfrom polymerizing. In addition, scavenging of free-radicals by thesolvent (e.g. via formation of hydroxy radicals and/or ethoxy radicals)may convert aromatic radicals into non-radical aromatics. This in turnmay reduce the potential for cross-linking involving aromatics in thebio-oil product.

Accordingly, processing of lignocellulosic matter in accordance with themethods of the invention circumvents a number of deficiencies associatedwith existing bio-fuel production methods and also provides a means ofexploiting the energy-producing potential of lignin.

Lignocellulosic Matter

The methods described herein are suitable for producing a bio-oilproduct from a material comprising lignin and cellulose. Any materialcomprising lignin and cellulose may be used. The material may compriseany number of substances in addition to lignin and cellulose.Alternatively, the material may consist predominantly of lignin andcellulose, or consist of lignin and cellulose only. In certainembodiments, material utilised in the methods of the inventionadditionally comprises proteins.

In certain embodiments, the material utilised in the methods of theinvention is lignocellulosic matter. In general, lignocellulosic matterrefers to a substance comprising the components of lignin, cellulose andhemicellulose.

The relative proportion of lignin, hemicellulose and cellulose in agiven sample will depend on the nature of the lignocellulosic matter.

For example, in some embodiments lignocellulosic matter used in themethods of the invention comprises 2-35% lignin, 15-45% cellulose and10-35% hemicellulose.

In other embodiments, lignocellulosic matter used in the methods of theinvention comprises 20-35% lignin, 20-45% cellulose and 20-35%hemicellulose.

In other embodiments, the content of lignin in the lignocellulosicmatter is more than 35%, or less than 20%, the content of cellulose ismore than 45% or less than 20%, and the content of hemicellulose is morethan 35% or less than 20%.

In some embodiments, the lignocellulosic matter comprises at least about10% lignin, at least about 15% cellulose, and at least about 10%hemicellulose.

In other embodiments, the lignocellulosic matter comprises at leastabout 15% lignin, at least about 20% cellulose, and at least about 15%hemicellulose.

In additional embodiments, the lignocellulosic matter comprises at leastabout 20% lignin, at least about 25% cellulose, and at least about 20%hemicellulose.

In some embodiments, the lignocellulosic matter comprises at least about25% lignin, at least about 30% cellulose, and at least about 25%hemicellulose.

The skilled addressee will recognize that the methods described hereinare not constrained by the relative proportions of lignin, hemicelluloseand cellulose in a given source of lignocellulosic matter.

Lignocellulosic matter for use in the methods of the invention may bederived from any source.

For example, woody plant matter may be used as a source oflignocellulosic matter.

Examples of suitable woody plants include, but are not limited to, pine(e.g. Pinus radiata), birch, eucalyptus, bamboo, beech, spruce, fir,cedar, poplar, willow and aspen. The woody plants may be coppiced woodyplants (e.g. coppiced willow, coppiced aspen).

By way of example only, the proportion of hemicellulose in woody plantmatter may be between about 15% and about 40%, the proportion ofcellulose may be between about 30% and about 60%, and the proportion oflignin may be between about 5% and about 40%. Preferably, the proportionof hemicellulose of the woody plant matter is between about 23% andabout 32%, the proportion of cellulose between about 38% and about 50%,and the proportion of lignin between about 15% and about 25%.Additionally or alternatively, fibrous plant matter may be used as asource of lignocellulosic matter, non-limiting examples of which includegrass (e.g. switchgrass), grass clippings, flax, corn cobs, corn stover,reed, bamboo, bagasse, hemp, sisal, jute, cannibas, hemp, straw, wheatstraw, abaca, cotton plant, kenaf, rice hulls, and coconut hair.

Suitable agricultural sources of lignocellulosic matter include, but arenot limited to, agricultural crops, crop residues, and grain processingfacility wastes (e.g. wheat/oat hulls, corn fines etc.). In general,agricultural source materials may include branches, bushes, canes, cornand cornhusks, energy crops, forests, fruits, flowers, grains, grasses,herbaceous crops, leaves, bark, needles, logs, roots, saplings, shortrotation woody crops, shrubs, switch grasses, trees, vines, hard woodsand soft woods.

Additionally or alternatively, lignocellulosic matter may be derivedfrom commercial or virgin forests (e.g. trees, saplings, scrap wood suchas branches, leaves, bark, logs, roots and products derived from theprocessing of such materials).

Additionally or alternatively, products and by-products comprisinglignocellulosic matter may be used as a source of lignocellulosicmatter. Non-limiting examples include wood-related materials and woodywastes (e.g. agricultural residue, forestry or timber processingresidue, waste or byproduct streams from wood products, sawmill andpaper mill discards and off-cuts, sawdust, particle board and leaves)and industrial products (e.g. pulp, paper, papermaking sludge,cardboard, textiles and cloths, dextran, and rayon).

Lignocellulosic matter may optionally be pre-treated prior to performingthe methods of the invention. For example, mechanical and/or chemicalmethods may be used to disrupt the structure of lignocellulosic matter.Non-limiting examples of mechanical pre-treatment methods includepressure, grinding, agitation, shredding, milling,compression/expansion, or other types of mechanical action.Pre-treatment of the lignocellulosic matter may be performed using amechanical apparatus, for example, an extruder, a pressurized vessel, ora batch reactor.

Pre-treatment methods may include treatment with heat. For example,steam explosion pre-treatment methods may be used to disrupt thestructure of lignocellulosic matter. In general, steam explosionpre-treatment methods involve exposing the matter to high pressure steamin a contained environment before the resulting product is explosivelydischarged to an atmospheric pressure. Pre-treatment with steamexplosion may additionally involve agitation of the lignocellulosicmatter.

In preferred embodiments, lignocellulosic matter for use in the methodsof the invention is provided in the form of a slurry. The slurry may begenerated, for example, by converting the lignocellulosic matter into apowder of appropriate particle size (e.g., by using grinding, agitation,shredding, milling, compression/expansion and/or other types ofmechanical action) and mixing with an appropriate liquid (e.g. water oraqueous alcohol).

The particle size of solid matter included in the slurry may be betweenabout 10 microns and about 10,000 microns. For example, the particlesize of solid matter included in the slurry may be at least about 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000 or 9000 microns. Alternatively,the particle size may be between about 10 microns and 50 microns,between about 10 microns and about 100 microns, between about 10 micronsand about 400 microns, between about 10 microns and about 500 microns,between about 100 microns and about 200 microns, between about 100microns and about 300 microns, between about 100 microns and about 500microns, or between about 100 microns and about 1000 microns.

In one embodiment, the particle size is between about 100 microns andabout 400 microns.

In another embodiment, the particle size is between about 50 microns andabout 500 microns.

In another embodiment, the solid matter is wood flour and the particlesize is between about 150 microns and about 300 microns.

The concentration of solid matter in the slurry may be high (e.g. aboveabout 50% w/v). Alternatively, the concentration of solid matter in theslurry may be between about 1% and about 50%, between about 1% and about40%, between about 1% and about 30%, between about 1% and about 20%, orbetween about 1% and about 10% w/v.

In certain embodiments, the concentration of solid matter in the slurryis between about 5% and about 20% w/v.

In one embodiment, the solid matter is wood flour and the concentrationof solid matter in the slurry is about 10% w/v.

In alternative embodiments, methods of the invention are conducted usinga material comprising lignin without cellulose. As used herein amaterial comprising lignin “without” cellulose will be understood toinclude a material with no cellulose but also a material comprising asmall amount of cellulose (as may be the case after purification orfractionation of lignin from a more complex material).

In another alternative embodiment, methods of the invention areconducted using a material comprising cellulose without lignin. As usedherein a material comprising cellulose “without” lignin will beunderstood to include a material with no lignin but also a materialcomprising a small amount of lignin (as may be the case afterpurification or fractionation of cellulose from a more complexmaterial).

One or more pre-treatment steps may be conducted to separate,up-concentrate and/or purify lignin and/or cellulose from a startingmaterial comprising additional substances.

Fractionation of Hemicellulose

The methods of the invention may be used to generate a bio-oil productfrom any material comprising lignin and cellulose.

In certain embodiments, the material is lignocellulosic matter. Inembodiments where the material is lignocellulosic matter, hemicellulosemay be fractionated prior to converting lignin and cellulose into abio-oil.

“Fractionation” of hemicellulose from lignocellulosic matter ascontemplated herein refers to a process whereby hemicellulose ispartially or wholly separated from other components (e.g. lignin and/orcellulose) of the same matter.

Following hemicellulose fractionation, the remaining solid mattercomprising predominantly lignin and cellulose may be treated with asolvent to produce a bio-oil product using the methods of the invention.

In alternative embodiments, the remaining solid matter may be separatedor substantially separated into lignin and cellulose components, eitheror both of which may be treated to produce an bio-oil product using themethods of the invention.

Lignocellulosic matter may optionally be pre-treated prior tohemicellulose fractionation, for example, as described in the sectionabove entitled “Fractionation of lignocellulosic matter”. Thefractionation of hemicellulose from lignocellulosic matter willgenerally involve the cleavage of specific chemical bonds. For example,covalent cross-linkages between hemicellulose and lignin may be brokento facilitate the fractionation. This may involve the cleavage of esterlinkages, for example, between the α-carbon of the phenylpropane subunitin lignin and the free carboxyl group of uronic acids and aromatic acidsin hemicellulose.

Additionally or alternatively, cleavage of ester linkages between theα-carbon of the phenylpropane subunit in lignin and hydroxyls inhemicellulose such as L-arabinose (O-5), D-glucose or D-mannose (O-6),O-2 xylose, O-3 xylose or glycosidic hydroxyl (O-1) may also occurduring fractionation of hemicellulose chains from lignin.

Fractionation of hemicellulose may also involve the cleavage of bondsexisting between hemicellulose and cellulose (e.g. hydrogen bonds)and/or bonds within the structure of hemicellulose (e.g. β(1→4) linkagesbetween monosaccharide units or α(1→6) side branch linkages).

Fractionation of hemicellulose in accordance with the methods of theinvention will generally involve the use of one or more solvents. Anysolvent capable of solvating hemicellulose can potentially be used,non-limiting examples of which include water, aqueous acidic solutions,aqueous alkaline solutions, and organic solvents. Suitable reactionconditions for the solvation of hemicellulose from lignocellulosicmatter will depend on the specific solvent or solvents used, and thenature of the lignocellulosic starting material.

Preferably, hemicellulose fractionation is conducted under mildconditions thereby minimizing sugar dehydration and the formation oftar-like molecules through polymerization.

In preferred embodiments, the hemicellulose is fractionated by solvationin aqueous solution. In general, solvation of hemicellulose in aqueoussolution will typically also involve partial hydrolysis of thehemicellulose. Examples of suitable aqueous solutions for the solvationand partial hydrolysis of hemicellulose include aqueous acidicsolutions, aqueous alkaline solutions, and aqueous solutions of neutralpH (i.e. pH of about 7.0).

A suitable alkaline aqueous solution may have a pH of above about 7.0,or above about 7.5. For example, a suitable alkaline aqueous solutionmay have a pH of between about 7.0 and about 11.0. In certainembodiments, the alkaline aqueous solution has a pH of between about 7.0and about 10.5, between about 8.0 and about 10.5, between about 7.0 andabout 10.0, between about 7,0 and about 9.5, between about 7.0 and about9.0, between about 7.0 and about 8.5, between about 7.0 and about 8.0,between about 7.2 and about 8.0, or between about 7.0 and 7.5.

A suitable acidic aqueous solution may have a pH of below about 7.0, orbelow about 6.5. For example, a suitable acidic aqueous solution mayhave a pH of between about 2.0 and about 7.0, or between about 3.0 andabout 7.0. In certain embodiments, the acidic aqueous solution has a pHof between about 3.5 and about 6.0, between about 3.5 and about 7.0,between about 4.0 and about 7.0, between about 4.5 and about 7.0,between about 5.0 and about 7.0, between about 5.5 and about 7.0,between about 6.0 and about 7.0, between about 6.0 and about 6.8, orbetween about 6.5 and about 7.0.

In one preferred embodiment, hemicellulose is fractionated fromlignocellulosic biomass in aqueous solution at neutral pH (i.e. pH 7.0)or substantially neutral pH.

In another preferred embodiment, hemicellulose is fractionated fromlignocellulosic biomass in aqueous solution at a pH of between about 6.5and about 7.5.

In another preferred embodiment, hemicellulose is fractionated fromlignocellulosic biomass in acidic aqueous solution at a pH of about 2.0.

In most cases, the pH of the reaction mixture can be adjusted by addinga suitable acid or base.

Non-limiting examples of suitable acids that may be used to adjust thepH of a reaction mixture include hydrochloric acid, trifluoroaceticacid, sulfuric acid, sulfurous acid and organic acids such as propionicacid, lactic acid, citric acid, or glycolic acid. Additionally oralternatively, carbon dioxide may be added to the reaction mixture toobtain an acidic pH (i.e. a pH of below about 7.0)

Non-limiting examples of suitable bases that may be used to adjust thepH of a reaction mixture include sodium hydroxide, potassium hydroxide,ammonium hydroxide, carbonates and bicarbonates.

Methods by which the pH of a reaction mix may be determined are known inthe art, and described, for example in Gallagher and Wiley (Eds) CurrentProtocols Essential Laboratory Techniques John Wiley & Sons, Inc (2008).

The solvation of hemicellulose in aqueous solution may be performed atany reaction temperature (in combination with any of the pH ranges orvalues referred to above). For example, the solvation of hemicellulosein aqueous solution may be performed at a reaction temperature ofbetween about 120° C. and about 250° C. In certain embodiments of theinvention, the reaction temperature is between about 130° C. and about250° C., between about 140° C. and about 250° C., between about 150° C.and about 250° C., between about 160° C. and about 250° C., betweenabout 170° C. and about 250° C., between about 180° C. and about 250°C., between about 190° C. and about 250° C., between about 200° C. andabout 250° C., between about 210° C. and about 250° C., between about220° C. and about 250° C., between about 230° C. and about 250° C.,between about 240° C. and about 250° C., between about 120° C. and about240° C., between about 120° C. and about 230° C., between about 120° C.and about 220° C., between about 120° C. and about 210° C., betweenabout 120° C. and about 200° C., between about 120° C. and about 190°C., between about 120° C. and about 180° C., between about 120° C. andabout 170° C., between about 120° C. and about 160° C., between about120° C. and about 150° C., between about 120° C. and about 140° C. orbetween about 120° C. and about 130° C.

In one preferred embodiment, hemicellulose is fractionated from thelignocellulosic matter at reaction temperatures ranging from about 120°C. to about 190° C.

Suitable reaction temperatures may be obtained, for example, byperforming the solvation of hemicellulose in a mechanical apparatus suchas a batch reactor or pressurized vessel. Performing the solvation ofhemicellulose in a mechanical apparatus, may also allow alteration ofthe pressure applied at the operating temperatures contemplated.

The solvation of hemicellulose in aqueous solution may be performed atany reaction pressure (in combination with any of the ranges/values ofreaction temperatures and/or reaction pH referred to above).

For example, the solvation of hemicellulose in aqueous solution may beperformed at a reaction pressure of between about 0.1 MPa (1 bar) andabout 25 MPa (250 bar), between about 0.1 MPa (1 bar) and about 10 MPa(100 bar), between about 0.1 MPa (1 bar) and about 5 MPa (50 bar),preferably between about 0.2 MPa (2 bar) and about 5 MPa (50 bar), andmore preferably between about 1 MPa (10 bar) and about 4 MPa (40 bar).

In a preferred embodiment, hemicellulose is fractionated fromlignocellulosic matter at a reaction pressure of between about 0.2 MPa(2 bar) and about 5 MPa (50 bar).

In another preferred embodiment, hemicellulose is fractionated fromlignocellulosic matter at a reaction pressure of between about 1 MPa (10bar) and about 4 MPa (40 bar).

In general, reactions are performed for a period of time sufficient tosolvate substantially all of the hemicellulose, or, the majority ofhemicellulose from the lignocellulosic matter.

For example, a reaction may be performed under conditions defined by acombination of any of the ranges/values of reaction temperature,reaction pressure and/or reaction pH referred to above for less than 20minutes. In some embodiments, the reaction is performed for betweenabout 2 minutes and about 20 minutes. In other embodiments, the reactionis performed from between about 5 minutes and about 15 minutes. In otherembodiments, the reaction is performed for a period of more than 20minutes.

Optimal reaction conditions for the solvation of hemicellulose fromlignocellulosic matter will ultimately depend on factors including thetype of lignocellulosic matter under treatment and the specific solventused. For example, factors such as temperature and pH of the reactionmixture, isotonicity, amount of lignocellulosic matter and solvent, andlength of reaction time may be varied in order to optimise the reaction.

Optimal reaction conditions will be readily apparent to the skilledaddressee upon analysis of the solvated hemicellulose, which may beperformed using standard methods generally known in the art. Forexample, solvated hemicellulose may be analysed using spectroscopytechniques. Suitable spectroscopy techniques include, but are notlimited to, near infra red spectroscopy, fourier transform infraredspectroscopy, nuclear magnetic resonance spectroscopy, raman microscopy,LTV microspectrophotometry and X-ray diffraction. Additionally oralternatively, solvated hemicellulose may quantified by high performanceliquid chromatography, for example, using methods described in Bjerre etal., “Quantification of solubilized hemicellulose from pretreatedlignocellulose by acid hydrolysis and high performance liquidchromatography”, (1996) in publication Riso-R-855 (EN), Rise NationalLaboratory.

In one preferred embodiment, hemicellulose is fractionated fromlignocellulosic matter at a reaction temperature of between about 100°C. and 250° C., and a reaction pressure of between about 0.2 MPa (2 bar)and about 5 MPa (50 bar). The pH of the reaction mix may be about 7.0,above about 7.0, or below about 7.0. The pH of the reaction mix may beabout 2.0.

In another preferred embodiment, hemicellulose is fractionated fromlignocellulosic matter at a reaction temperature of between about 100°C. and 250° C., and a reaction pressure of between about 1 MPa (10 bar)and about 4 MPa (40 bar). The pH of the reaction mix may be about 7.0,above about 7.0, or below about 7.0. The pH of the reaction mix may beabout 2.0.

In another preferred embodiment, the hemicellulose component isfractionated from the lignocellulosic matter using water at a reactionpH of about 7.0 and a reaction temperature of about 210° C.

In certain embodiments of the invention hemicellulose is fractionatedfrom lignocellulosic matter by solvation with a sub-critical solvent. Inthe context of the present specification, a sub-critical solvent is afluid at a temperature and pressure below its thermodynamic criticalpoint.

In one embodiment, hemicellulose is solvated using sub-critical water.For example, sub-critical water may be used at temperature of less thanabout 374° C. and a pressure of less than about 22.1 MPa (221 bar).Suitable reaction temperatures and pressures may be facilitated, forexample, by performing the solvation of hemicellulose in a batchreactor, a pressurized vessel or an autoclave.

In certain embodiments, the solvation of hemicellulose in sub-criticalwater may be performed at a reaction temperature of between about 100°C. and about 270° C. In other embodiments, the reaction temperature isbetween about 120° C. and about 270° C., between about 140° C. and about270° C., between about 160° C. and about 270° C., between about 180° C.and about 270° C., between about 200° C. and about 270° C., betweenabout 220° C. and about 270° C., between about 240° C. and about 270°C., between about 260° C. and about 270° C., between about 100° C. andabout 250° C., between about 100° C. and about 230° C., between about100° C. and about 210° C., between about 100° C. and about 190° C.,between about 100° C. and about 170° C., between about 100° C. and about150° C., or between about 100° C. and about 130° C.

Solvation of hemicellulose in sub-critical water performed at any of theabove-mentioned temperatures may be performed, for example, at apressure of less than about 22 MPa (220 bar), less than about 20 MPa(200 bar), less than about 16 MPa (160 bar), less than about 12 MPa (120bar), less than about 8 MPa (80 bar), less than about 4 MPa (40 bar),less than about 3 MPa (30 bar), less than about 2 MPa (20 bar), or about1 MPa (10 bar).

In one embodiment, hemicellulose is fractionated from lignocellulosicmatter by solvation in sub-critical water at a temperature of about 190°C. and a pressure of about 3 MPa (30 bar).

The solvated hemicellulose component may be removed from the remainingsolid matter (which substantially comprises lignin and cellulose) usingany suitable means. For example, remaining solid matter comprisinglignin and cellulose may be physically retained by passing the mixturethrough one or more appropriately sized filters through which thesolvated hemicellulose fraction may pass. The solid matter may beretained on the filter(s) and washed if so desired.

Additionally or alternatively, centrifugation may be used to separatesolvated hemicellulose from remaining solid matter. In a continuoussystem, counter current flow of solids and liquid may be used tofacilitate the separation.

In certain embodiments, a hydrocyclone apparatus is used to separate thesolvated hemicellulose fraction from the remaining matter comprisinglignin and cellulose. A hydrocyclone is a static apparatus that appliescentrifugal force to a liquid mixture so as to promote the separation ofheavy components, in this case the remaining solid matter, from lightcomponents, in this case the solvated hemicellulose fraction. Ingeneral, a hydrocyclone may operate to separate hemicellulose fromremaining solid matter as follows. The hydrocyclone directs inflowtangentially near the top of a vertical cylinder, converting thevelocity of incoming material into a rotary motion thus creatingcentrifugal force. The remaining solid matter moves outward toward thewall of the cylinder where it agglomerates and spirals down the wall toan outlet. The solvated hemicellulose fraction moves toward the axis ofthe hydrocyclone and upwards to a different outlet.

Following hemicellulose fractionation, the remaining biomass comprisingpredominantly lignin and cellulose may be treated with a solvent toproduce a bio-oil product using the methods of the invention.

Alternatively, the remaining biomass may be fractionated into lignin andcellulose components, either or both of which may be treated to producea bio-oil product using the methods of the invention.

Bio-Oil Production from Cellulose and Lignin

The methods of the invention provide a means of generating a bio-oilproduct from material comprising lignin and cellulose using a solventunder defined reaction conditions. In general, the bio-oil product isstable. The bio-oil product may be in the form of an emulsion.

Without being limited to a particular mechanism or mode of action, it isbelieved that a solvent used in accordance with the methods of theinvention facilitates mechanical swelling of the lignin and cellulosepresent in the material under treatment. This may be responsible for anumber of effects including, for example, assisting “opening up” of thesubstrate making it more accessible and prone to hydrolysis anddepolymerization. In addition, the swelling may in itself disrupthydrogen bonds in the substrate (e.g. those present between celluloseand lignin).

For example, in the case where an aqueous alcohol (e.g. aqueous ethanolor aqueous methanol) is utilized to generate a bio-oil product inaccordance with the methods of the invention, it is thought that thealcohol is able to penetrate the lignin/cellulose composite as it isless polar than water. Under certain reaction conditions water isbelieved to dissolve organic substances such as hydrocarbons and thusmay also interact closely with the substrate to facilitate swelling.Solvation of the substrate is thought to be facilitated, at least inpart, by solvent-mediated hydrolysis (e.g. base and acid catalysis). Forexample, hydrolysis of carbohydrates may occur predominantly through thehydrolysis of glycosidic linkages, while hydrolysis of lignin (i.e.lignin depolymerization) may be facilitated by ether linkage hydrolysis(where the ether contains at least one aromatic). In addition, it isthought that dehydration of the carbohydrates may lead to theelimination of water and formation of double bonds.

In general, the solvation of lignin is believed to arise at least inpart from the cleavage of chemical bonds within the branched structureof lignin, such as ether or carbon-carbon linkages. Specific examples oflinkages in the structure of lignin that may be cleaved include, but arenot limited, to β-O-4 linkages (e.g. phenylpropane β-aryl ether), 5-5linkages (e.g. biphenyl and dibenzodioxocin), β-5 linkages (e.g.phenylcoumaran), β-1 linkages (e.g. 1,2-diaryl propane), α-O-4 linkages(e.g. phenylpropane α-aryl ether), 4-O-5 linkages (e.g. diaryl ether)and β-β linkages (e.g. β-β-linked structures). The solvation ofcellulose is believed to arise at least in part from the chemical bondsincluding, for example, β-1,4-linkages between D-glucose units.Solvation may additionally involve the cleavage of bonds existingbetween lignin and cellulose (e.g. hydrogen bonds and ether linkages).

It is also postulated that a solvent used in accordance with the methodsof the invention may act as a chemical stabilization agent. Againwithout being limited to a particular mechanism or mode of action,stabilization may occur through various interactions with both reactionintermediates and the bio-oil product. Chemical stabilization may beaffected, for example, by alkylation, arylation, interaction withphenolic groups and/or free radical scavenging. In general, chemicalstabilization serves to prevent cross-linking and polymerization, eventswhich are believed to yield tar-like compounds. In addition, scavengingof free-radicals by the solvent (e.g. via formation of hydroxyl radicalsand/or ethoxy radicals) may have the effect of converting aromaticradicals into non-radical aromatics. This in turn may reduce thepotential for cross-linking involving aromatics in the bio-oil product.

In accordance with the methods of the invention, the conversion ofmaterial comprising lignin and cellulose into a bio-oil product isconducted using a solvent at elevated temperatures. Again without beingbound to a particular mechanism or mode of action, it is believed thatthe elevated temperatures facilitate decarboxylation and elimination(dehydration) reactions whereby much of the oxygen contained in thebiomass is removed as carbon dioxide gas and water, respectively.

Any material comprising lignin and cellulose may be used to perform themethods of the invention. The material may comprise any number ofsubstances in addition to lignin and cellulose. Alternatively, thematerial may consist predominantly of lignin and cellulose, or consistof lignin and cellulose only.

In preferred embodiments, the material is lignocellulosic matter or isderived from lignocellulosic matter.

In alternative embodiments, the methods are used to generate bio-oilfrom a material comprising lignin from which cellulose has beencompletely or substantially removed (as may be the case afterpurification or fractionation of lignin from a more complex material).

In other alternative embodiments, the methods are used to generatebio-oil from a material comprising cellulose from which lignin has beencompletely or substantially removed (as may be the case afterpurification or fractionation of cellulose from a more complexmaterial).

The methods for bio-oil production provided herein generally involvetreatment of a material comprising lignin and cellulose with a solvent.When the material is lignocellulosic matter, it is contemplated thathemicellulose will first be fractionated and removed prior to generationof the bio-oil from lignin/cellulose. Preferably, the hemicellulose isfractionated and removed using the methods described above in thesection entitled “Fractionation of hemicellulose”.

In preferred embodiments of the invention, bio-oil is generated frommaterial comprising lignin and cellulose matter provided in the form ofa slurry. The slurry may be formed, for example, by reducing the matterinto a powder of appropriate particle size (e.g. by using grinding,agitation, shredding, milling, compression/expansion and/or other typesof mechanical action) and mixing with an appropriate liquid (e.g. anaqueous solvent).

In certain embodiments, the slurry is formed from solid mattercomprising lignin and cellulose remaining after the fractionation ofhemicellulose from lignocellulosic matter (for example, as described inthe section above entitled “Fractionation of lignocellulosic matter”).

The particle size of solid matter included in the slurry may be betweenabout 10 microns and about 10,000 microns. For example, the particlesize of solid matter included in the slurry may be at least about 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000 or 9000 microns. Alternatively,the particle size may be between about 10 microns and about 50 microns,between about 10 microns and about 100 microns, between about 10 micronsand about 400 microns, between about 10 microns and about 500 microns,between about 100 microns and about 200 microns, between about 100microns and about 300 microns, between about 100 microns and about 500microns between about 100 microns and about 500 microns, or betweenabout 100 microns and about 1000 microns.

In one embodiment, the particle size is between about 100 microns andabout 400 microns.

In another embodiment, the particle size is between about 50 microns andabout 500 microns.

In another embodiment, the particle size is between about 150 micronsand about 300 microns.

The concentration of solid matter in the slurry may be above about 50%w/v. Alternatively, the concentration of solid matter in the slurry maybe between about 1% and about 50%, between about 1% and about 40%,between about 1% and about 30%, between about 1% and about 20%, orbetween about 1% and about 10% w/v.

The concentration of solid matter in the slurry may be about 5%, about10%, about 15%, about 20%, about 25% or about 30% w/v.

In certain embodiments, the concentration of solid matter in the slurryis between about 4% and about 30% w/v.

In certain embodiments, the slurry comprises between about 2% and about45% solid matter by weight.

In certain embodiments, the slurry comprises between about 2% and about30% solid matter by weight.

In certain embodiments, the slurry comprises about 5% solid matter byweight.

Any solvent capable of generating a bio-oil product from materialcomprising lignin and/or cellulose under the reaction conditionsdescribed herein may be used. The solvent may be utilised undersupercritical conditions, sub-critical conditions or at conditions whichoscillate above and below the thermodynamic critical point of thesolvent.

In preferred embodiments, the solvent is an aqueous solvent (e.g. anaqueous acidic solution, an aqueous alkaline solution, or an aqueoussolution of neutral pH (i.e. pH of about 7.0)). In the context of thepresent specification, an “aqueous solvent” is a solvent containing atleast one percent water based on total weight of solvent. The ratio ofsolvent to water may be above about 0.01 (i.e. 1 part solvent: 99 partswater). Preferably, the ratio of solvent to water is equal to or aboveabout 0.11 (i.e. 1 part solvent: 9 parts water). More preferably, theratio of solvent to water is equal to or above about 0.25 (i.e. 1 partsolvent: 4 parts water). The aqueous solvent may comprise water andbetween about 1% and 40% weight solvent.

In certain embodiments the solvent is an alkylating agent. Thealkylating agent will, in general, comprise an alkyl chain bearing anappropriate leaving group. The transfer of an alkyl chain from thealkylating agent to the lignin/cellulose composite may facilitatesolvation and/or chemical stabilization of the composite.

Non-limiting examples of suitable alkylating agents includealkylhalides, alkylsulfates, olefins, alkylphosphates, and alcohols.

Non-limiting examples of alkylhalides include methyl chloride, isopropylchloride, ethyl bromide, and methyl iodide.

Non-limiting examples of alkylaromatics include xylenes, andtrimethylbenzenes.

Non-limiting examples of suitable olefins include monoolefins such asethylene, propylene, n-butene, isobutylene, 1-pentene, 1-hexene,cyclohexene, and 1-octene.

A non-limiting example of a suitable diolefin is 1,3-Butadiene.

Preferably, alcohol (e.g. an aqueous alcohol) is used as a solvent forthe bio-oil production methods described herein. Suitable alcohols mayhave between about one and about ten carbon atoms. Non-limiting examplesof preferred alcohols include methanol, ethanol, isopropyl alcohol,isobutyl alcohol, pentyl alcohol, hexanol and iso-hexanol.

In certain embodiments, the aqueous alcohol comprises between about 1%and about 30% alcohol by weight.

In certain embodiments, the aqueous alcohol comprises between about 5%and about 30% alcohol by weight.

In certain embodiments, the aqueous alcohol comprises about 25% alcoholby weight.

In certain embodiments, the aqueous alcohol comprises about 20% alcoholby weight.

In certain embodiments, the solvent comprises a mixture of aqueousalcohols (e.g. an aqueous mixture comprising methanol and at least oneother alcohol, an aqueous mixture comprising ethanol and at least oneother alcohol, an aqueous mixture comprising methanol and ethanol, or anaqueous mixture comprising methanol and ethanol and at least one otheralcohol).

In certain embodiments, the solvent comprises a mixture of aqueousalcohols comprising between about 5% and about 30% alcohol by weight,comprising between about 5% and about 30% alcohol by weight, comprisingabout 25% alcohol by weight, or comprising about 20% alcohol by weight.

In preferred embodiments of the invention, the solvent used to producebio-oil from material comprising lignin and/or cellulose is ethanol.

In particularly preferred embodiments, the ethanol is aqueous ethanol.The ratio of ethanol to water may be equal to or above about 0.01 (i.e.1 part ethanol: 99 parts water). Preferably, the ratio of ethanol towater is equal to or above about 0.11 (i.e. 1 part ethanol: 9 partswater). More preferably, the ratio of ethanol to water is equal to orabove about 0.25 (i.e. 1 part alcohol: 4 parts water).

In certain embodiments, the aqueous ethanol comprises between about 1%and about 30% ethanol by weight.

In certain embodiments, the aqueous ethanol comprises between about 5%and about 30% ethanol by weight.

In certain embodiments, the aqueous alcohol comprises about 25% ethanolby weight.

In certain embodiments, the aqueous alcohol comprises about 20% ethanolby weight.

Using the methods of the invention, materials comprising lignin andcellulose may be converted into a bio-oil product using a solvent (forexample, any one or more of the specific alcohols, aqueous alcohols, ormixtures of aqueous alcohols referred to above) at a reactiontemperature or a range of reaction temperatures of between about 200° C.and about 400° C., or between about 250° C. and about 400° C. In certainembodiments, the reaction temperature or range of reaction temperaturesis between about 230° C. and about 360° C., between about 230° C. andabout 350° C., between about 230° C. and about 340° C., between about230° C. and about 330° C., between about 230° C. and about 320° C.,between about 230° C. and about 310° C., between about 230° C. and about300° C., between about 230° C. and about 290° C., between about 230° C.and about 280° C., between about 230° C. and about 270° C., betweenabout 230° C. and about 260° C., between about 230° C. and about 250°C., between about 230° C. and about 240° C., between about 230° C. andabout 350° C., between about 240° C. and about 350° C., between about250° C. and about 350° C., between about 260° C. and about 350° C.,between about 270° C. and about 350° C., between about 280° C. and about350° C., between about 290° C. and about 350° C., between about 300° C.and about 350° C., between about 310° C. and about 350° C., betweenabout 320° C. and about 350° C., between about 330° C. and about 350°C., or between about 340° C. and about 350° C. In certain embodiments,the reaction temperature is 320° C.

Using the methods of the invention, any of the above-mentioned reactiontemperatures or ranges of reaction temperatures may be combined with areaction pressure or a range of reaction pressures of between about 10MPa (100 bar) and about 30 MPa (300 bar), between about 12 MPa (120 bar)and about 24 MPa (240 bar), between about 14 MPa (140 bar) and about 24MPa (240 bar), between about 15 MPa (150 bar) and about 24 MPa (240bar), between about 16 MPa (160 bar) and about 24 MPa (240 bar), betweenabout 17 MPa (170 bar) and about 24 MPa (240 bar), between about 18 MPa(180 bar) and about 24 MPa (240 bar), between about 19 MPa (190 bar) andabout 24 MPa (240 bar), between about 20 MPa (200 bar) and about 24 MPa(240 bar), between about 21 MPa (210 bar) and about 24 MPa (240 bar),between about 22 MPa (220 bar) and about 24 MPa (240 bar), between about23 MPa (230 bar) and about 24 MPa (240 bar), between about 12 MPa (120bar) and about 22 MPa (220 bar), between about 12 MPa (120 bar) andabout 18 MPa (180 bar), between about 12 MPa (120 bar) and about 16 MPa(160 bar), between about 12 MPa (120 bar) and about 14 MPa (140 bar),between about 14 MPa (140 bar) and about 23 MPa (230 bar), between about14 MPa (140 bar) and about 22 MPa (220 bar), between about 14 MPa (140bar) and about 21 MPa (210 bar), between about 14 MPa (140 bar) andabout 20 MPa (200 bar), between about 14 MPa (140 bar) and about 19 MPa(190 bar), between about 14 MPa (140 bar) and about 18 MPa (180 bar),between about 14 MPa (140 bar) and about 17 MPa (170 bar), between about14 MPa (140 bar) and about 16 MPa (160 bar), between about 14 MPa (140bar) and about 15 MPa (150 bar), or about 20 Mpa (200 bar).

Using the methods of the invention, conversion of matter comprisinglignin and cellulose to a bio-oil may be performed using a combinationof any of the above-mentioned reaction temperatures/ranges of reactiontemperatures and reaction pressures/ranges of reaction pressures at asuitable reaction pH. For example, the pH may be neutral, acidic (i.e.less than 7.0) or basic (i.e. more than 7.0). In certain embodiments,the pH is between about 6.5 and 7.5.

In general, reactions to produce bio-oil in accordance with theinvention are performed for a period of time sufficient to convertsubstantially all of the lignin and cellulose in the material, or, themajority of lignin and cellulose in the material to a bio-oil. Forexample, a reaction defined by any combination of the values/ranges ofvalues of temperature, pressure and/or pH set forth above may beperformed for a period of between 2 minutes and 60 minutes. In someembodiments, the reaction is performed for between about 2 minutes andabout 40 minutes. In some embodiments, the reaction is performed forbetween about 5 minutes and about 40 minutes. In other embodiments, thereaction is performed from between about 5 minutes and about 30 minutes.In other embodiments, the reaction is performed for a period of lessthan about 20 minutes.

Specific reaction conditions utilized for the methods of bio-oilproduction provided herein will depend on factors such as the type ofsolvent used, whether the solvent is aqueous and if so the percentage ofwater in the solvent, the amount of starting material, the specific typeof starting material and so on. For example, factors such as temperatureand pH of the reaction mixture, isotonicity, amount of startingmaterial, amount of solvent, and length of reaction time may be variedin order to optimize the reaction.

The solvent composition (e.g. percentage of water if aqueous) andtemperature/pressure utilized during the reaction can be optimized so asto maximize the yield and/or reduce the processing time. In preferredembodiments, all or substantially all of the lignin and cellulose in agiven starting material is converted into the bio-oil product.

Desired reaction conditions may be achieved, for example, by conductingthe reaction in a suitable mechanical apparatus capable of maintainingincreased temperature and/or increased pressure. A suitable mechanicalapparatus will, in general, include any apparatus provided with suitableheating means that is designed to generate and withstand pressure.

It will be understood that a solvent used to produce a bio-oil inaccordance with the methods of the invention may do so under conditionsof temperature and pressure that are above the critical point of thesolvent (i.e. supercritical), below the critical point of the solvent(i.e. sub-critical) and/or at the critical point of the solvent. Thecritical point of a solvent used in the methods will depend on factorssuch as the percentage of water (if an aqueous solvent is used) and thechemical state of the material under treatment. For example, thecritical point of a given solvent is likely to change over the course ofa given reaction as input material becomes solvated. It is alsoenvisaged that reaction conditions in accordance with the methods of theinvention may oscillate around the critical point of a substance (i.e.movement from supercritical conditions to sub-critical conditions andvice versa).

In certain embodiments, material comprising lignin and cellulose (e.g. aslurry comprising 2% to 45% solid matter by weight) is converted into abio-oil product using aqueous alcohol as the solvent (e.g. any of thespecific aqueous ethanol solvents referred to above) at a reactiontemperature or a range of reaction temperatures of between about 250° C.and 400° C., and a reaction pressure or a range of reaction pressures ofbetween about 10 MPa (100 bar) and about 25 MPa (250 bar), for a periodof between about 2 minutes and about 60 minutes. Preferably, the aqueousalcohol is aqueous ethanol, Preferably, the aqueous ethanol comprisesbetween about 1% and about 30% ethanol by weight and more preferablybetween about 5% and about 30% ethanol by weight. Still more preferably,the aqueous ethanol comprises about 20% or about 25% ethanol by weight.

In other embodiments, material comprising lignin and cellulose (e.g. aslurry comprising 2% to 30% solid matter by weight) is converted into abio-oil product using aqueous ethanol comprising between about 15% andabout 30% ethanol by weight, at a reaction temperature or a range ofreaction temperatures of between about 280° C. and 350° C., and areaction pressure or a range of reaction pressures of between about 15MPa (150 bar) and about 25 MPa (250 bar), for a period of between about5 minutes and about 30 minutes.

In further embodiments, material comprising lignin and cellulose (e.g. aslurry comprising 2% to 30% solid matter by weight) is converted into abio-oil product using aqueous ethanol comprising between about 20% andabout 25% ethanol by weight, at a reaction temperature or a range ofreaction temperatures of between about 280° C. and 330° C., a reactionpressure or a range of reaction pressures of between about 18 MPa (180bar) and about 22 MPa (220 bar), for a period of between about 5 minutesand about 20 minutes.

In other embodiments, material comprising lignin and cellulose (e.g. aslurry comprising 4% to 30% solid matter by weight) is converted into abio-oil product using aqueous ethanol comprising between about 20% andabout 25% ethanol by weight, at a reaction temperature or a range ofreaction temperatures of between about 280° C. and 330° C., and areaction pressure or a range of reaction pressures of between about 18MPa (180 bar) and about 22 MPa (220 bar), for a period of between about5 minutes and about 20 minutes.

In one embodiment, a bio-oil product is formed from a materialcomprising lignin and cellulose using aqueous ethanol (1 part ethanol:99 parts water) at a reaction temperature of about 320° C. and areaction pressure of about 18 MPa (180 bar).

In one embodiment, a bio-oil product is formed from a materialcomprising lignin and cellulose using aqueous ethanol (1 part ethanol: 9parts water) at a reaction temperature of about 320° C. and a reactionpressure of about 18 MPa (180 bar).

In another embodiment, a bio-oil product is formed from a materialcomprising lignin and cellulose using aqueous ethanol (1 part ethanol: 4parts water) at a reaction temperature of about 320° C. and a reactionpressure of about 18 MPa (180 bar).

Bio-Oil Production from Cellulose

In alternative embodiments of the invention, a bio-oil product isgenerated using a material comprising cellulose (i.e. cellulosicmaterial) from which lignin has been completely or substantially removed(as may be the case after purification or fractionation of cellulosefrom a more complex material). Bio-oil may be generated from thematerial using any of the methods (including reaction conditions)described in the section above entitled “Bio-oil production fromcellulose and lignin”.

Lignocellulosic matter may be used to produce cellulosic material fromwhich lignin has been completely or substantially removed.

For example, cellulosic material from which lignin has been completelyor substantially removed may be obtained by fractionating lignin (andoptionally hemicellulose) from lignocellulosic matter, as described inthe section below entitled “Bio-oil production from lignin”.

Alternatively, the cellulosic material may be generated by fractionatingcellulose from lignocellulosic matter. In preferred embodiments, thefractionation is performed after an initial step of hemicellulosefractionation as described in the section above entitled “Fractionationof hemicellulose”.

Fractionation of cellulose from lignocellulosic matter may be achievedusing a solvent.

Examples of suitable solvents and methods by which cellulose may besolvated are described in U.S. Pat. No. 2,179,181, U.S. Pat. No.3,447,939, U.S. Pat. No. 4,097,666, U.S. Pat. No. 4,302,252, U.S. Pat.No. 5,410,034, and U.S. Pat. No. 6,824,599.

Examples of methods by which cellulose may be solvated includehydrolytic disintegration by use of superheated steam at elevatedpressure. Additionally or alternatively, cellulose may be solvated usingionic liquids, or tertiary amines.

Solvents suitable for fractionating cellulose from lignocellulosicmatter or modified forms thereof (e.g. lignocellulosic matter withhemicellulose removed or substantially removed) include, but are notlimited to, water, aqueous acidic solutions, aqueous alkaline solutions,and organic solvents.

Preferably, cellulose is fractionated from lignocellulosic matter or amodified form thereof using an aqueous solvent. In general,fractionation of cellulose by solvation in aqueous solution will alsoinvolve partial hydrolysis of the cellulose.

The aqueous solvent may be an aqueous acidic solvent, an aqueous basicsolvent, or an aqueous solvent of neutral pH (i.e. pH of about 7.0). Asuitable basic aqueous solution will have a pH of greater than about7.0. For example, a suitable basic aqueous solvent may have a pH ofbetween about 7.0 and about 12.0. A suitable acidic aqueous solvent mayhave a pH of less than about 7.0. For example, a suitable acidic aqueoussolvent may have a pH of between about 7.0 and about 2.0.

The solvation of cellulose in an aqueous solvent may be performed at anysuitable reaction temperature (in combination with any of the ranges orvalues of pH referred to above).

For example, the reaction temperature may be between about 80° C. andabout 400° C. In certain embodiments of the invention, the reactiontemperature is between about 100° C. and about 400° C., between about120° C. and about 400° C., between about 140° C. and about 400° C.,between about 160° C. and about 400° C., between about 180° C. and about400° C., between about 200° C. and about 400° C., between about 220° C.and about 400° C., between about 240° C. and about 400° C., betweenabout 260° C. and about 400° C., between about 280° C. and about 400°C., between about 300° C. and about 400° C., between about 320° C. andabout 400° C., between about 340° C. and about 400° C., between about360° C. and about 400° C., between about 380° C. and about 400° C.,between about 80° C. and about 380° C., between about 80° C. and about360° C., between about 80° C. and about 340° C., between about 80° C.and about 320° C., between about 80° C. and about 300° C., between about80° C. and about 280° C., between about 80° C. and about 260° C.,between about 80° C. and about 240° C., between about 80° C. and about220° C., between about 80° C. and about 200° C., between about 80° C.and about 180° C., between about 80° C. and about 160° C., between about80° C. and about 140° C., between about 80° C. and about 120° C.,between about 80° C. and about 100° C., or between about 80° C. andabout 90° C.

In one embodiment, the cellulose is solvated and partially hydrolysedusing water at a pH of about 7.0 and a reaction temperature of about340° C.

The solvation of cellulose in aqueous solution may be performed at anyreaction pressure (in combination with any of the ranges or values ofreaction temperature and/or reaction pH referred to above).

For example, the solvation of cellulose in aqueous solution may beperformed at a reaction pressure of between about 0.01 MPa (0.1 bar) andabout 25 MPa (250 bar), between about 0.01 MPa (0.1 bar) and about 10MPa (100 bar), between about 0.01 MPa (0.1 bar) and about 5 MPa (50bar), preferably between about 0.02 MPa (0.2 bar) and about 5 MPa (50bar) and more preferably between about 1 MPa (10 bar) and about 4 MPa(40 bar).

In general, reactions are performed for a period of time sufficient tosolvate (i.e. fractionate) substantially all of the cellulose, or, themajority of cellulose.

For example, a reaction under conditions defined by a combination of anyof the values or ranges of reaction pH and/or reaction temperatureand/or reaction pressure referred to above may be performed for lessthan 20 minutes. In some embodiments, the reaction is performed forbetween about 2 minutes and about 20 minutes. In other embodiments, thereaction is performed from between about 5 minutes and about 15 minutes.In other embodiments, the reaction is performed for a period of morethan 20 minutes.

Optimal reaction conditions for the solvation of cellulose willultimately depend on factors including the purity of the cellulose typeunder treatment and the specific solvent used. For example, factors suchas temperature and pH of the reaction mixture, isotonicity, amount ofcellulosic matter and solvent, and length of reaction time may be variedin order to optimise the reaction.

Optimal reaction conditions will be readily apparent to the skilledaddressee upon analysis of the solvated cellulose, which may beperformed using standard methods generally known in the art. Forexample, solvated cellulose may be analysed using spectroscopytechniques. Suitable spectroscopy techniques include, but are notlimited to, near infra red spectroscopy, fourier transform infraredspectroscopy, nuclear magnetic resonance spectroscopy, raman microscopy,UV microspectrophotometry and X-ray diffraction. Additionally oralternatively, solubilized cellulose may quantified by high performanceliquid chromatography.

In certain embodiments, the fractionation of cellulose fromlignocellulosic matter may be achieved by treatment with supercriticalwater. In general, water may be brought into a supercritical state byheating to above a temperature of about 370° C. under pressure of about22.0 MPa (220 bar).

Supercritical conditions may be achieved, for example, by conducting thereaction in a suitable mechanical apparatus capable of maintainingincreased temperature and/or increased pressure. Examples of a suitablemechanical apparatus include an autoclave, a supercritical reactor, orany apparatus provided with suitable heating means and designed towithstand the pressures utilized. In general, the apparatus willpreferably provide a means of mixing a solvent with the materialcomprising cellulose and bringing/maintaining the solvent in the mixtureto a supercritical state.

Cellulosic material from which lignin has been completely orsubstantially removed may be further treated or modified prior toconversion to a bio-oil using the methods of the invention. This may bedone to assist or enhance the chemical or physical characteristics ofthe cellulose-containing material such that it is better suited for oilconversion using the methods described herein.

Bio-Oil Production from Lignin

In alternative embodiments of the invention, a bio-oil product isgenerated using a material comprising lignin from which cellulose hasbeen completely or substantially removed (as may be the case afterpurification or fractionation of lignin from a more complex material).Bio-oil may be generated from the material using any of the methods(including reaction conditions) described in the section above entitled“Bio-oil production from cellulose and lignin”.

Material comprising lignin from which cellulose has been completely orsubstantially removed may be obtained by fractionating cellulose (andoptionally hemicellulose) from lignocellulosic matter, as described inthe section below above “Bio-oil production from cellulose”.

Alternatively, the material may be generated by fractionating ligninfrom lignocellulosic matter. In preferred embodiments, the fractionationis performed after an initial step of hemicellulose fractionation asdescribed in the section above entitled “Fractionation ofhemicellulose”.

Fractionation of lignin from lignocellulosic matter may be achieved, forexample, by treatment with a supercritical solvent. In preferredembodiments, the fractionation is performed after an initial step ofhemicellulose fractionation as described in the section above entitled“Fractionation of hemicellulose”.

In general, a supercritical solvent is a solvent heated above itscritical temperature and pressurized above its critical pressure suchthat it exhibits properties of both a gas and a liquid. However, it willbe understood that the term “supercritical” as used herein alsoencompasses conditions of temperature and/or pressure that are a small,although not substantial, amount (e.g. approximately 5%) below thesupercritical point of the substance in question (i.e. “sub-critical”).Accordingly, the term “supercritical” also encompasses oscillatorybehaviour around the supercritical point of a substance (i.e, movementfrom supercritical conditions to sub-critical conditions and viceversa).

Any supercritical solvent may be used that is capable of solvatinglignin from biomass. Non-limiting examples of suitable solvents includenitrous oxide, sulfur dioxide, ammonia based solvents, amines, carbondioxide, and mixtures thereof.

Fractionation of lignin with a supercritical solvent may be performed ata temperature that is at least the critical temperature for the solventselected, and preferably, above the critical temperature. When suchoperating temperatures are contemplated, the pressure applied during thereaction will be at least equivalent to that required to maintain thesolvent as a supercritical fluid. Temperature, solvent composition, andpressure range during the solvation of lignin can be selected so as tomaximize lignin fractionation as well as to decrease processing time.Examples of supercritical temperatures and pressures for varioussolvents suitable for the solvation of lignin are provided in Table 1below,

TABLE 1 non-limiting examples of various supercritical solvents that maybe utilised to solvate lignin from lignocellulosic matter (or a modifiedform thereof with hemicellulose removed) Molecular Critical CriticalCritical weight temperature pressure density Solvent g/mol K MPa (atm)g/cm³ Carbon dioxide 44.01 304.1 7.38 (72.8) 0.469 (CO₂) Water (H₂O)18.02 647.3 22.12 (218.3) 0.348 Methane (CH₄) 16.04 190.4 4.60 (45.4)0.162 Ethane (C₂H₆) 30.07 305.3 4.87 (48.1) 0.203 Propane (C₃H₈) 44.09369.8 4.25 (41.9) 0.217 Ethylene 28.05 282.4 5.04 (49.7) 0.215 (C₂H₄)Propylene 42.08 364.9 4.60 (45.4) 0.232 (C₃H₆) Methanol 32.04 512.6 8.09(79.8) 0.272 (CH₃OH) Ethanol 46.07 513.9 6.14 (60.6) 0.276 (C₂H₅OH)

Supercritical conditions may be achieved, for example, by conducting thereaction in a suitable mechanical apparatus capable of maintainingincreased temperature and/or increased pressure. Examples of a suitablemechanical apparatus include an autoclave, a supercritical reactor, orany apparatus provided with suitable heating means and that is designedto withstand the pressures utilized. In general, the apparatus willpreferably provide a means of mixing a solvent with the materialcomprising lignin and bringing/maintaining the solvent in the mixture toa supercritical state.

In one embodiment of the invention, a supercritical alcohol is used tosolvate the lignin component. Examples of suitable alcohols include, butare not limited to, methanol, ethanol, isopropyl alcohol, isobutylalcohol, pentyl alcohol, hexanol and iso-hexanol.

In a preferred embodiment, lignin is fractionated from biomass usingsupercritical ethanol. In general, ethanol may be brought into asupercritical state by heating the reaction above a temperature of aboveabout 245° C. under pressure of above about 6.0 MPa (60 bar).

In certain embodiments, lignin is separated from solid matter remainingafter fractionation of hemicellulose from lignocellulosic matter. Theseparation of lignin is performed using supercritical ethanol as asolvent at a reaction temperature of above about 230° C. and a pressureof above about 5.5 MPa (55 bar). Preferably, the reaction is performedat a reaction temperature of above about 250° C. and a pressure of aboveabout 6.5 MPa (65 bar). In certain embodiments, the reaction isperformed for between about 2 minutes and about 15 minutes. Preferablythe reaction is performed for between about 3 minutes and about 10minutes.

The solvated lignin fraction may be removed from remaining solid matter,for example, by using cyclone apparatus. A cyclone apparatus may operateto separate lignin from remaining solid matter as follows. A high speedrotating air-flow comprising solvated lignin may be established within aconical or cylindrical cyclone, the air flowing in a spiral pattern froman upper (wider) end to a lower (narrower) end. The air flow exits thecyclone in a straight stream through the center of the cyclone and outthe upper portion. Particles of remaining solid matter in the rotatingair stream have too much inertia to remain in the air stream, and fallto the bottom of lower end of the cyclone where they are removed.

Material comprising lignin from which cellulose has been completely orsubstantially removed may be further treated or modified prior toconversion to oil using the methods described herein. This may be doneto assist or enhance the chemical or physical characteristics of thelignin-containing material such that it is better suited for oilconversion using the methods described herein.

Bio-Oil Product

Certain embodiments of the invention relate to a bio-oil productobtained or obtainable by the methods of the invention. The bio-oilproduct will, in general, be a stable bio-oil product.

The bio-oil product may comprise compounds including, but not limitedto, linear and branched aliphatics and aromatics with and withoutfunctional groups (e.g. hexane, toluene), methoxyphenol,ethylmethoxyphenol and methoxypropenylphenol. Compounds within thebio-oil may comprise functional groups including, but not limited to,phenols (e.g. ArOH), aldehydes (e.g. RCHO), aromatic groups, alkylatinggroups (e.g. olefin), oxygen-containing functional groups (e.g.alcohols, ethers, aldehydes, ketones, and carboxylic acids), methyl,methylene and aromatic methyl.

The bio-oil product may be produced in the form of an emulsion.Non-limiting examples of compounds that may be present in the emulsioninclude phenol, 2-cyclopentene-1-one, 2-methyl, methoxyphenol,ethylmethoxyphenol, and methoxypropylphenol.

In certain embodiments, the emulsion comprises a lighter aqueous phaseand heavier black oil phase.

The lighter aqueous phase may comprise compounds including, but notlimited to, Ether,1-propenylpropyl, 2-Cyclopenten-1-one, 2-methyl-,Phenol, Phenol, 2-methoxy-, 2,3-Dimethylhydroquinone, Phenol,4-ethyl-2-methoxy-, 1,2-Benzenediol, 4-methyl-, Phenol,2-methoxy-4-propyl-, Vanillin, and Phenol, 2-methoxy-.

The heavier black oil phase may comprise about 70%-80% carbon, and about5%-10% hydrogen. The black oil phase may comprise compounds including,but not limited to, Phenol, 4-ethyl-2-methoxy-, Phenol,2-methoxy-4-propyl-, Oleic Acid, 2-Isopropyl-10-methylphenanthrene,3-(3-Hydroxy-4-methoxyphenyl)-1-alanine, (−)-Nortrachelogenin,7-(3,4-Methylenedioxy)-tetrahydrobenzofuranone, 1-Phenanthrenecarboxylicacid, 1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-,methyl ester, [1R-(1.alpha, 4a.beta.,10a.alpha)],1-Phenanthrenecarboxylic acid,1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-,[1R-(1.alpha., 4a.beta.,10a.alpha)], and Carinol.

The bio-oil phase may be separated from the emulsion using standardtechniques known in the art, examples of which include the use of hightemperatures, pressure, gravity, microfiltration, chemicals (e.g. suchas extractants and demulsifiers), high shear, and sonic energy. Specificexamples of methods by which oil may be separated from the emulsioninclude the use of high shear or turbulence to drive the oil from themixture (see for example, U.S. Pat. No. 4,481,130), devices such asthose described in U.S. Pat. No. 5,538,628 and U.S. Pat. No. 4,483,695,and processes such as those described in PCT publication No. WO2001/074468.

Preferably, the bio-oil product has an energy content of between about10 MJ/kg and about 30 MJ/Kg. In certain embodiments, the bio-oil producthas an energy content of between about 10 MJ/kg and about 25MJ/Kg,between about 18 MJ/kg and about 28MJ/Kg, or between about 10 MJ/kg andabout 15MJ/Kg. In specific embodiments, the bio-oil product has anenergy content of about 30 MJ/Kg.

The bio-oil product may be used in any number of applications. Incertain embodiments, the bio-oil is used as a biofuel. The bio-oilproduct may be used directly. Additionally or alternatively, the bio-oilmay be used as a fuel additive. For example, the bio-oil product may beblended with other fuels, including for example, ethanol, biodiesel andthe like. Additionally or alternatively, the bio-oil product may befurther processed, for example, for conversion into another fuel.

Saccharification and Fermentation of Hemicellulose

Fractionated hemicellulose obtained in accordance with the methodsdescribed of the invention may be subjected to saccharification toproduce fermentable sugars. For example, saccharification offractionated hemicellulose may produce polysaccharides,oligosaccharides, disaccharides, monosaccharides or mixtures thereof.Preferably, saccharification of the hemicellulose component will producepolysaccharide chains comprising between about two and about 50monosaccharide units. More preferably, saccharification of thehemicellulose component will produce polysaccharide chains comprisingbetween about two and about 10 monosaccharide units, and/or betweenabout five monosaccharide units and about two monosaccharide units. Mostpreferably, saccharification of the hemicellulose component will producemonosaccharides.

Production of shorter polysaccharide chains, oligosaccharides,disaccharides and/or monosaccharides may be achieved by the cleavage ofone or more chemical bonds present in fractionated hemicellulose usingany suitable means. Non-limiting examples of preferred bonds within thestructure of hemicellulose that may be cleaved include S-glycosidicbonds, N-glycosidic bonds, C-glycosidic bonds, O-glycosidic bonds,α-glycosidic bonds, β-glycosidic bonds, 1,2-glycosidic bonds,1,3-glycosidic bonds, 1,4-glycosidic bonds and 1,6-glycosidic bonds,ether bonds, hydrogen bonds and/or ester bonds.

Saccharification of fractionated hemicellulose may be performed usingany suitable method known in the art.

For example, pyrolysis may be used to cleave chemical bonds infractionated hemicellulose to produce shorter polysaccharides,oligosaccharides, disaccharides, monosaccharides or mixtures thereof. Ingeneral, pyrolysis involves cleavage of chemical bonds by theapplication of heat. Non-limiting examples of pyrolysis techniques thatmay be utilized for saccharification include anhydrous pyrolysis(performed in the absence of oxygen), hydrous pyrolysis (performed inthe presence of water) and vacuum pyrolysis (performed in a vacuum).Methods by which heat may be provided for pyrolysis are generally knownin the art and include, for example, direct heat transfer using a hotgas or circulating solids and indirect heat transfer with exchangesurfaces such as walls or tubes. Suitable reactors for pyrolysis aredescribed, for example, in U.S. Pat. No. 3,853,498, U.S. Pat. No.4,510,021, Scott et al., Canadian Journal of Chemical Engineering (1984)62: 404-412 and Scott et al., Industrial and Engineering ChemistryProcess and Development (1985) 24: 581-588.

Additionally or alternatively, saccharification of fractionatedhemicellulose may be achieved by hydrolysis. For example, fractionatedhemicellulose may be hydrolyzed by the addition of a dilute acid (e.g.sulfuric acid), a dilute base, or pH neutral water with the applicationof heat.

Hemicellulose fractionated from lignocellulosic matter may be hydrolyzedusing one or more hydrolytic enzymes. Any enzyme capable of catalyzingthe hydrolysis of hemicellulose to produce shorter polysaccharides,oligosaccharides, disaccharides, monosaccharides and mixtures thereofmay be used. In general, hydrolytic enzymes suitable forsaccharification of hemicellulose fractionated using the methods of theinvention are those classified under EC 3 (hydrolases) of the enzymenomenclature of the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (NC-IUBMB)(http://www.chem.qmul.ac.uk/iubmb/) nomenclature as of the filing dateof this application. Preferably, the hydrolytic enzymes utilized arethose classified under class EC 3.2 (glycosylases) of the NC-IUBMBenzyme nomenclature.

In certain embodiments, hydrolytic enzymes suitable for use in themethods described herein are those classified under subclass 3.2.1(Glycosidases, i.e. enzymes hydrolyzing O- and S-glycosyl compounds) ofthe NC-IUBMB nomenclature. In other embodiments, hydrolytic enzymes thatmay be utilized are those classified under subclass EC 3.2.2(Hydrolyzing N-Glycosyl Compounds) of the NC-IUBMB nomenclature. Inother embodiments, hydrolytic enzymes that may be utilized are thoseclassified under subclass EC 3.2.3 (Hydrolyzing S-Glycosyl Compounds) ofthe NC-IUBMB nomenclature.

Non-limiting examples of glycoside hydrolases and carbohydrases suitablefor use in the methods described herein and commercial sources of thoseenzymes are described in US Patent Publication No. 20060073193.Preferred examples include cellulases, xylanases, arabinosidases,β-glucosidases, β-xylosidases, mannanases, galactanases, dextranases,endoglucanases, and alpha-galactosidase.

Hydrolytic enzymes may be applied in a purified or substantiallypurified form to the fractionated hemicellulose, or in combination withother substances or compounds (e.g. as part of a culture supernatant).Additionally or alternatively, a hydrolytic enzyme-producingmicroorganism or mixtures of microorganisms capable of producinghydrolytic enzymes may be cultured in the presence of hemicellulosefractionated in accordance with the methods described herein to providea source of hydrolytic enzymes.

Hydrolytic enzymes suitable for use in accordance with the methodsdescribed herein may be derived from any suitable microorganism,including but not limited to, bacteria and fungi/yeast. Themicroorganism may be a psychrophilic, mesophilic, thermophilic orextremely thermophilic organism, in accordance with the classificationdescribed in Brock, 1986, “Thermophiles: General Molecular and AppliedMicrobiology”, (T. D Brock, Ed) John Wiley and Sons, Inc. New York, andBergquist et al., 1987, Biotechnol Genet. Eng. Rev. 5:199-244.

In one embodiment, enzymatic hydrolysis of fractionated hemicellulose isperformed using thermophilic hydrolytic enzymes. The use of thermostablehydrolytic enzymes for the hydrolysis of fractionated hemicelluloseoffers several advantages over the use of hydrolytic enzymes thatoperate optimally at lower temperatures, including higher specificactivity and higher stability. Typically, thermophilic hydrolyticenzymes display hydrolytic activity at elevated reaction temperatures.For example, a thermophilic hydrolytic enzyme will typically remainactive at a reaction temperature of more than 60° C.

Non-limiting examples of bacteria from which suitable hydrolytic enzymesmay be derived include Acidothermus sp. (e.g. A. cellulolyticus),Anaerocellum sp. (e.g. A. thermophilum), Bacillus sp., Butyrivibrio sp.(e.g. B. fibrisolvens), Cellulomonas sp. (e.g. C. fimi), Clostridium sp.(e.g. C. thermocellum, C. stercorarium), Erwinia sp. (e.g. E.chrysanthemi), Fibrobacter sp. (e.g. F. succinogenes), Micromonosporasp., Rhodothermus sp. (e.g. R. marinus), Ruminococcus sp. (e.g. R.albus, R. flavefaciens), Streptomyces sp., Thermotoga sp. (e.g. T.maritima, T. neapolitana), Xanthomonas sp. (e.g. X. campestris) andZymomonas sp. (e.g. Z. mobilis).

Non-limiting examples of fungi/yeast from which suitable hydrolyticenzymes may be derived include Aureobasidium sp., Aspergillus sp. (e.g.A. awamori, A. niger and A. oryzae), Candida sp., Chaetomium sp. (e.g.C. thermophilum, C. thermophila), Chrysosporium sp. (e.g. C.lucknowense), Corynascus sp. (e.g. C. thermophilus), Dictyoglomus sp.(e.g. D. thermophilum), Emericella sp., Fusarium sp., Gliocladium sp.,Hansenula sp., Humicola sp. (e.g. H. insolens and H. grisea), Hypocreasp., Kluyveromyces sp., Myceliophthera sp. (e.g. M. thermophila),Neurospora sp., Penicillium sp., Pichia sp., Rhizomucor sp. (e.g. R.pusillus), Saccharomyces sp., Schizosaccharomyces sp., Sporotrichum sp.,Thermoanaerobacterium sp. (e.g. T. saccharolyticum), Thermoascus sp.(e.g. T. aurantiacus, T. lanuginosa), Thermomyces sp. (e.g. T.lanuginosa), Thermonospora sp. (e.g. T. curvata, T. fusca), Thielaviasp. (e.g. T. terrestris), Trichoderma sp. (e.g. T. reesei, T. viride, T.koningii, T. harzianum), and Yarrowia sp.

Suitable microorganisms that naturally produce hydrolytic enzymes, forexample any of the bacteria or fungi/yeasts referred to above, may becultured under suitable conditions for propagation and/or expression ofthe hydrolytic enzyme or enzymes of interest. Methods and conditionssuitable for the culture of microorganisms are generally known in theart and are described in, for example, Current Protocols in Microbiology(Coico et al. (Eds), John Wiley and Sons, Inc, 2007).

Recombinant organisms may be used as a source of hydrolytic enzymes forsaccharification of hemicellulose fractionated in accordance with themethods described herein. Additionally or alternatively, recombinantorganisms capable of producing hydrolytic enzymes may be cultured withfractionated hemicellulose. Recombinant microorganisms includingbacterial or fungal/yeast strains expressing one or more hydrolyticenzymes derived from an exogenous source may be generated. Methods forthe production of recombinant microorganisms are generally known in theart and are described, for example, in Ausubel et al., (Eds) CurrentProtocols in Molecular Biology (2007) John Wiley & Sons; Sambrook etal., Molecular Cloning: A Laboratory Manual, (2000) 3rd Ed., Cold SpringHarbor Laboratory Press; Molecular Cloning (Maniatis et al., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1982); and CurrentProtocols in Microbiology (Coico et al. (Eds), John Wiley and Sons, Inc,2007).

The reaction conditions for enzymatic hydrolysis are typically based onconsideration of the conditions suitable for the specific enzyme ormixture of enzymes. In general, typical conditions for enzymatichydrolysis include a reaction temperature of between about 30° C. andabout 90° C., and a pH of between about 4.0 and about 8.0. Suitablereaction temperatures and pH for enzymatic hydrolysis of polysaccharidesare described, for example, in Viikari et al., “Thermostable Enzymes inLignocellulosic Hydrolysis”, 2007, 108:121-145.

Non-limiting examples of oligosaccharide fragments that may be producedby saccharification of hemicellulose include oligosaccharides such asmannan-oligosaccharides, fructo-oligosaccharides andgalacto-oligosaccharides.

Non-limiting examples of disaccharide fragments that may be produced bysaccharification of hemicellulose include sucrose, lactose, maltose,trehalose, cellobiose, laminaribiose, xylobiose, gentiobiose,isomaltose, mannobiose, kojibiose, rutinose, nigerose, and melibiose.

Non-limiting examples of monosaccharide fragments that may be producedby saccharification of hemicellulose include trioses includingaldotrioses (e.g. glyceraldehyde) and ketotrioses (e.g.dihydroxyacetone), tetroses including aldotetroses (e.g. threose anderythrose) and ketotetroses (e.g. erythrulose), pentoses includingaldopentoses (e.g. lyxose, ribose, arabinose, deoxyribose) andketopentoses (e.g. xylulose and ribulose), hexoses including aldohexoses(e.g. glucose, mannose, altrose, idose, galactose, allose, talsoe andgulose) and ketohexoses (e.g. fructose, psicose, tagatose and sorbose),heptoses including keto-heptoses (e.g. sedoheptulose andmannoheptulose), octoses including octolose and2-keto-3-deoxy-manno-octonate and nonoses including sialose.

In a preferred embodiment, saccharification of the hemicellulosefractions yields an aqueous solution comprising shorter lengthpolysaccharide chains, oligosaccharides, disaccharides, monosaccharides,or mixtures thereof.

In an alternative embodiment of the invention, fractionatedhemicellulose obtained in accordance with the methods described hereinmay be subjected to hydrothermal upgrading in sub-supercritical water toproduce fermentable sugars. Methods for hydrothermal upgrading are knownin the art and are described for example in Srokol et al., “Hydrothermalupgrading of biomass to biofuel; studies on some monosaccharide modelcompounds” Carbohydr Res. 2004 Jul. 12; 339(10):1717-26.

Certain embodiments of the invention relate to saccharides obtainable orobtained from fractionated hemicellulose in accordance with the methodsdescribed herein.

In accordance with the methods described herein, sugars derived fromfractionated hemicellulose may be fermented to produce one or morefermented sugar products. For example, the microorganism may be capableof converting saccharide fragments into alcohols (e.g. ethanol), ororganic acids (for example succinic acid and glutamic acid). The organicacids may be used in the production of other products, for examplebiopolymers, amino acids and antibiotics. Suitable microorganisms forfermentation include, but are not limited to, bacteria, fungi/yeast,and/or recombinant varieties of those organisms.

Fermentation may be performed directly on fractionated hemicellulose.Additionally or alternatively, fermentation may be performed onfragmented saccharides derived from saccharification of the fractionatedhemicellulose. Additionally or alternatively, fermentation may beperformed simultaneously with saccharification of fractionatedhemicellulose. For example, a reaction mixture comprising hydrolyticenzymes and/or microorganisms capable of producing hydrolytic enzymesmay be combined with microorganisms that ferment sugars and appliedunder suitable culture conditions to hemicellulose fractionated inaccordance with the methods described herein.

In certain embodiments, residual lignin may be removed from thefractionated hemicellulose components prior to fermentation. Residuallignin may be removed, for example, using methods described in Mosier etal., “Features of promising technologies for pretreatment oflignocellulosic biomass”, 2005, Bioresource Technology, 96:673-86.

In general, fermentation may be performed using any microorganismcapable of converting saccharides into one or more desired fermentedsugar products. For example, the microorganism may be capable ofconverting saccharides into alcohols (including ethanol), or organicacids (for example succinic acid and glutamic acid). The organic acidsmay be used in the production of other fermented sugar products, forexample biopolymers, amino acids and antibiotics.

In certain embodiments, the microorganism is capable of fermentingsaccharides derived from fractionated hemicellulose into one or morealcohols. Non-limiting examples of alcohols that may be produced inaccordance with the methods described herein include xylitol, mannitol,arabinol, butanol and ethanol.

In a preferred embodiment, 5-carbon saccharides (pentoses) derived fromsaccharification of the hemicellulose fraction are fermented to producealcohols, non-limiting examples of which include xylitol, mannitol,arbinol and ethanol.

Non-limiting examples of microorganisms capable of producing ethanolfrom saccharides include Zymomonas sp. (e.g. Z. mobilis), Saccharomycessp. (e.g. S. cerevisiae), Candida sp. (e.g. C. shehatae),Schizosaccharomyces sp. (e.g. S. pombe), Pachysolen sp. (e.g. P.tannophilus), and Pichia sp. (e.g. P. stipitis).

Microorganisms suitable for the fermentation of saccharides to producemannitol include, for example, fungi/yeast and lactic acid bacteria.Suitable microorganisms will in general express enzymes necessary formannitol production, for example, mannitol dehydrogenase.

Examples of bacterial species that may be used for the fermentation ofsaccharides to mannitol include Leuconostoc sp. (e.g. Leuconostocmesenteroides), Lactobacillus sp. (e.g. L. bevis, L. buchnei, L.fermeyitum, L. sanfranciscensis), Oenococcus sp. (e.g. O. oeni),Leuconostoc sp. (e.g. L. mesenteriode) and Mycobacterium sp. (e.g. M.smegmatis).

Examples of fungi/yeast suitable for the fermentation of saccharides toproduce mannitol include, but are not limited to, Basidiomycetes sp.,Trichocladium sp., Geotrichum sp., Fusarium sp., Mucor sp. (e.g. M.rouxii), Aspergillus sp. (e.g. A. nidulans), Penicillium sp. (e.g. P.scabrosum), Candida sp. (e.g. C. zeylannoide, C. lipolitica),Cryptococcus sp. (e.g. C. neoformans) and Torulopsis sp. (e.g. T.mannitofaciens).

Methods for the fermentation of saccharides to produce mannitol aredescribed, for example, in U.S. Pat. No. 6,528,290 and PCT publicationNo. WO/2006/044608.

Microorganisms suitable for the fermentation of saccharides to producexylitol include yeasts such as Saccharomyces Candida sp. (e.g. C.magnoliae, C. tropicalis, C. guilliermondif), Pichia sp., andDebaryomyces sp. (e.g. D. hansenii). Methods for the fermentation ofxylitol from saccharides are described, for example, in U.S. Pat. No.5,081,026, U.S. Pat. No. 5,686,277, U.S. Pat. No. 5,998,181, and U.S.Pat. No. 6,893,849.

In preferred embodiments of the invention, fermentation of saccharidesis performed using one or more recombinant microorganisms. Methods forthe production of recombinant microorganisms are generally known in theart and are described, for example, in Ausubel et al., (Eds) CurrentProtocols in Molecular Biology (2007) John Wiley & Sons and Sambrook etal., Molecular Cloning: A Laboratory Manual, (2000) 3rd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. In general,recombinant microorganisms suitable for use in the methods describedherein will express one or more genes encoding enzymes necessary for theconversion of saccharides to the desired target product.

Examples of preferred recombinant ethanologenic microorganisms are thosewhich express alcohol dehydrogenase and pyruvate decarboxylase. Genesencoding alcohol dehydrogenase and pyruvate decarboxylase may beobtained, for example, from Zymomonas mobilis. Examples of recombinantmicroorganisms expressing one or both of these enzymes and methods fortheir generation are described, for example, in U.S. Pat. No. 5,000,000,U.S. Pat. No. 5,028,539, U.S. Pat. No. 424,202, and U.S. Pat. No.5,482,846.

Suitable recombinant microorganisms may be capable of converting bothpentoses and hexoses to ethanol. Recombinant microorganisms capable ofconverting pentoses and hexoses to ethanol are described, for example,in U.S. Pat. No. 5,000,000, U.S. Pat. No. 5,028,539, U.S. Pat. No.5,424,202, U.S. Pat. No. 5,482,846, and U.S. Pat. No. 5,514,583.

Culture conditions suitable for the fermentation of saccharides toalcohols, organic acids and other fermented sugar products are generallyknown in the art, and are described in, for example, Bonifacino et al.,(Eds) Current Protocols in Cell Biology (2007) John Wiley and Sons, Inc.and Coico et al., (Eds) Current Protocols in Microbiology (2007) JohnWiley and Sons, Inc. Generally, microorganisms may be cultured at atemperature of between about 30° C. and about 40° C., and a pH ofbetween about 5.0 and about 7.0. In may be advantageous to add cofactorsfor the fermenting enzymes and/or nutrients for the microorganisms tooptimize the enzymatic fermentation. For example, cofactors such asNADPH and/or NAD may be added to the culture to assist the activity offermenting enzymes (e.g. xylose reductase and xylitol dehydrogenase).Carbon, nitrogen and sulfur sources may also be included in the culture.

Fermented sugar products derived from fractionated hemicellulose may befurther refined or processed.

Accordingly, certain embodiments of the invention relate to fermentedsugar products obtained or obtainable from fractionated hemicelluloseproduced in accordance with the methods described herein.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

EXAMPLES

The invention will now be described with reference to specific examples,which should not be construed as in any way limiting

Example 1 Overview

A flow diagram illustrating certain embodiments of the invention isprovided below.

Example 2 Extraction of Hemicellulose from Wood Flour Wood Flour SlurryPreparation

A measured amount of water was added to a feed tank using a flowindicator. Wood flour was added to the tank and a stirrer used tosuspend the wood flour and form a slurry.

The slurry was fed into the plant reactor via a variable speed positivedisplacement pump. The speed of the pump was set to provide the requiredproduction rate. The pump may be fed with either wood flour slurry ortown water for startup and shutdown purposes via the automatic three wayvalve.

A pressure relief valve fitted to the discharge of the slurry feed pumplimited the maximum system pressure to 60 bar. The discharge pressurewas monitored by an online pressure transmitter. The feed tank may bedrained and flushed by diverting the discharge of the feed pump to drainvia a three way manual valve.

Heating

The slurry was heated in two stages, first using double or concentricpipe heat exchanger banks supplied with saturated steam from a boilerand subsequently with electric heating elements.

In the first heating stage the slurry was raised close to thetemperature of saturated steam (180° C.) fed directly from the boiler atfull pressure. The temperature exiting this heating state was monitoredby an online temperature transmitter. The steam condensate from thefirst heating stage was returned to the boiler water feed tank via steamtraps. This increased the thermal efficiency of the boiler and enabled agreater rate of steam generation.

Upon exiting the steam heating stage, the slurry made its way past three4 kW electric 3 m long heating elements clamped in series to the processtubing. This long heating path (9 m) gradually heated the slurry to thefinal target temperature (210° C.). The final temperature of the slurrywas monitored by an online temperature transmitter, and was controlledby varying the supply voltage to all three heating elements.

The heating stages were arranged to enable a slow and gradual heating ofthe slurry to the target temperature of 210° C., to avoid the risk ofthermally decomposing any material and leading to process blockages.

Reaction

Following heating to the target temperature, the slurry was retained ina series of larger diameter pipes (50 mm) for 5 minutes to providesufficient time for the reaction to occur. Residence time in the reactorcan be reduced to 2.5 minutes (if desired) by reconfiguring the reactorpiping. These reaction pipes were well insulated but not heated, and theexit temperature was monitored by an online temperature transmitter. Theplant was preheated prior to operation by running on water until thetarget conditions were reached.

Cooling

Upon exiting the reactor, the slurry was cooled to approximately 80° C.using a bank of concentric pipe heat exchangers and town water. Coolingto that temperature was required for operation of the vacuum filter, dueto the high vapour pressure of hot water preventing vacuum operation. Ingeneral, it is desirable to filter the slurry as hot as possible toreduce the risk of precipitation and deposit formation.

The exit temperature of the cooler was monitored by an onlinetemperature transmitter, and controlled by manipulating the flow of townwater with a control valve.

Filtering

After cooling, the slurry was discharged into the vat of a small vacuumdrum filter through a control valve. This control valve was used to setthe system pressure as monitored by the pressure transmitter. A threeway automatic valve also allowed alternate discharge through a manualvalve as a backup.

The slurry may also be discharged to drain by a three way automaticvalve. This enables the whole system to be started on water or flushedout at the end of a run. This valve also allows the plant to keepoperating for a short period should any problems arise with the filter.

The rotary drum filter included a vacuum pump and centrifugal pump.These collected and transferred the filtrate to and from a standpipefitted with a level switch. The filtrate (hemicellulose liquor/sugar)from the standpipe was discharged into a collection tank for later use.The synthetic filter cloth had an air permeability of approximately35cfm and covered an area of 1858 cm².

The drum filter used a rocking agitator to prevent sedimentation of theslurry in the collection vat. The drum and agitator drive motors werecontrolled by locally operated variable speed drives, to enable the cakethickness and filter performance to be optimized.

The filter cake was removed from the synthetic cloth covered drum by anadjustable doctor blade, from which it fell under gravity into a waterfilled receival tank constantly stirred by an agitator to break up andsuspend the filter cake. Prior to operation, that tank was filled withwater via a flow indicator to a level above the stirrer preventingpossible damage to the stirrer.

From the receival tank, the slurry (containing lignin and cellulose) wastransferred over to a feed tank in a different reactor (for furtherprocessing) by an air operated diaphragm pump, the air to which iscontrolled by a solenoid valve.

Shower System

A shower bar installed above the drum was used to wash the product(hemicellulose liquor/sugar) solution from the filter cake to maximizerecovery. The shower bar received hot town water from the cooler,reducing water consumption and enabling more effective washing overusing cold water.

The flow of wash water was controlled by a control valve and an onlineflow meter. The control valve diverted the excess hot water not requiredby the shower bar to drain.

The flow of wash water was controlled to ratio of the feed rate, so thatthe flow was sufficient to displace the bound liquor in the filter cakeonly. In this manner, good washing was achieved without excess dilutionof the product liquor.

The liberated hemicellulose may then undergo enzymatic de-polymerizationand subsequent fermentation and distillation by established methods toproduce ethanol. The remaining cellulose/lignin wood fractions can becollected as a solid and further treated.

EQUIPMENT SPECIFICATIONS Plant Reactor Specifications:

The fundamental plant reactor operating specifications were as follows:Feed Rate: 120 kg/hr of slurry per hourFeed Consistency Maximum of 10% dry solidsFeed Size Maximum particle size 300 micronsProcess Operating Pressure: 40 bar (gauge)Process Design Pressure: 60 bar (gauge)

Process Design Temperature: 250° C. Process Operating Pressure: 210° C.Heat Exchanger Jacket Design Pressure: 20 bar Heat Exchanger JacketDesign Temperature: 325° C.

Saturated Steam Delivery Pressure/Temperature: 10 bar (gauge); 180° C.

Example 3 Fractionation of Hemicellulose Liquor from Radiata Pine (Pinusradiata)

A series of different runs were performed in which hemicellulose liquorwas extracted from Radiata pine (Pinus radiata). Different reactionconditions used for each of thirteen representative runs are describedin Table 2 below.

Wood flour was prepared (150-300 microns) and combined with water in abatch tank to produce a slurry (5%-10% v/v solids concentration) whichwas then pumped into a reactor. The slurry was steam-heated to atemperature of 120° C.-210° C. and hemicellulose extracted at neutralPH, or under acidic conditions afforded by the addition of sulphuricacid (0.1%-0.4% wt) or carbon dioxide. Hemicellulose extractionreactions were performed for up to 10 minutes.

Upon completion of the reaction the mixture was passed through a filterto provide separate solid (lignin and cellulose) and liquid(hemicellulose and water) fractions. In some cases the solid fraction(filter cake) was washed to obtain residual hemicellulose liquor. Theseparated hemicellulose fraction was then analysed for sugar content asdescribed in Examples 4 and 5 below.

TABLE 2 Reaction conditions for hemicellulose extraction from P. radiataAvailable Run Conditions Variable range 1 2 3 4 5 6 Pressure (bar) 22-6040 40 40 30 30 30 Temperature 120-210 120-210 120-210 120-210 120-190120-190 120-190 (° C.) Solids     5-15% 10 10 10 10 20 10 Concentration(%) Retention  0-10 10 5 0 10 20 10 Time (min) pH 2-7 7 7 7 ~2 7 ~2Additives Ethanol, None None None 0.4% None Carbon phosphoric sulphuricdioxide acid, acid sulphuric acid, carbon dioxide Wood Flour 180 and 300300 300 300 300 300 Grade 300 micron Wood Species Radiata RadiataRadiata Radiata Radiata Radiata Radiata pine, Oak pine pine pine pinepine pine Available Run Conditions Variable range 7 8 9 10 11 12Pressure (bar) 22-60 30 30 30 30 30 30 Temperature 120-210 120-190160-190 160-190 190 140-160 190 (° C.) Solids     5-15% 15% or 10 10 1010 10 concentration greater (%) Retention  0-10 20 2.5 5 5 5 5 Time(min) pH 2-7 7 7 7 7 7 7 Additives Ethanol, None None None None 0.1% wtNone phosphoric sulphuric acid, acid sulphuric acid, carbon dioxide WoodFlour 180 and 300 300 300 300 150 300 150 Grade micron Wood SpeciesRadiata Radiata Radiata Radiata Radiata Radiata Radiata pine, Oak pinepine pine pine pine pine

Example 4 Production of Reducing Sugars from Hemicellulose FractionUsing Enzyme Hydrolysis

Enzyme hydrolysis was conducted on hemicellulose liquor fractionsobtained from Radiata pine samples by the process described in Example 3above.

3.1 Materials and Methods

Conditions for enzyme hydrolysis were as shown in Table 3 below.

TABLE 3 Enzymatic hydrolysis of samples pH Temp. pH (with duringExtractives Sample (liquor enzyme + sampling weight* Description/ Numbersamples) buffer) (° C.) (mg/mL) Comments 1.1 4.90 5.16 RT 5.2 10% FS 1.23.82 4.8 190 12.0 1.3 3.84 4.8 190 11.12 1.4 4.12 4.96 150 11.68 1.54.24 5.02 150 11.08 1.6 4.37 5.11 130 7.76 1.7 4.49 5.10 130 7.40 2.14.75 5.15 RT 5.16 10% FS 2.2 4.19 5.0 190 13.12 2.3 4.21 5.0 190 13.082.4 4.40 5.08 163 9.56 2.5 4.46 5.12 163 8.44 2.6 4.72 5.17 105 6.28 2.74.62 5.20 105 5.68 FS feedstock slurry; RT room temperature *based ondry weight from 25 mL of clear liquor samples dried in petri dish at 70°C., 14.5 hours

Buffers and pH

120 mM of universal buffer (pH 6.5) was included in reaction mixes toprovide optimal conditions for hydrolytic enzymes to act onhemicellulose present in the different fractions. The target pH duringthese assays was ˜5-6. As shown in Table 3 above, the pH of each samplewas measured before and after the addition of buffer and enzyme samples.

Hydrolytic Enzymes

A recombinant Trichoderma reesei strain was used to produce a mixture ofhydrolytic enzymes comprising both hydrolytic fungal enzymes and athermophilic xylanase (XynB).

Reaction Mixes

Reaction mixes for enzyme hydrolysis were prepared as follows:

(i) Hemicellulose liquor samples Substrate 500 μL Enzyme 300 μL Univ.buffer (pH 6.5) 200 μL (ii) Substrate only control Substrate 500 μLUniv. buffer (pH 6.5) 200 μL H₂O 300 μL (iii) Enzyme only control:Enzyme 300 μL Univ. buffer (pH 6.5) 200 μL H₂O 500 μL

All tubes were incubated at 50° C. (with rotation) for 1.5 hours thenremoved and kept at 4° C.

Colorimetric Reducing Sugar Assay

A colourimetric dinitrosalicyclic acid (DNS)-reducing sugar assay wasused as an indicator of enzyme hydrolysis (see Bailey and Poutanen(1989), “Production of xylanases by strains of Aspergillus”, Appl.Microbiol. Biotechnol. 30: 5-10), The DNS reducing sugar method testsfor the presence of free carbonyl groups (C═O), present on reducingsugars (e.g. glucose, xylose, mannose, etc). As a result,3,5-dinitrosalicyclic acid (DNS) is reduced to 3-amino,5-nitrosalicyclicacid under alkaline conditions and an intense orange-brown colour isformed, indicative of reducing sugars, etc.

50 μL of sample was collected from each tube after enzyme hydrolysis,mixed with 75 μL DNS and boiled for 5 minutes. Absorbance I₅₄₀ was readfrom 100 μL samples.

3.2 Results Colorimetric Reducing Sugar Assay

Absorbance readings obtained from the 100 μL samples labelled 1.1-1.7and 2.1-2.7 were plotted and are shown in FIG. 1. These results areindicative of the presence and subsequent increase in reducing endsfollowing hydrolysis with the mixture of hydrolytic enzymes utilised.

Example 5 Total Sugar-Acid Hydrolysis and Analysis by High PerformanceLiquid Chromatography (HPLC)

Enzyme hydrolysis was conducted on hemicellulose liquor fractionsobtained from Radiata pine samples by the process described in Example 3above.

4.1 Materials and Methods

Total sugar analysis of was performed according to the Standard TestMethod for Carbohydrate Distribution of Cellulosic Materials, TAPPIStandard, Designation: D 5896-96 (2007), with some minor modifications.

In brief, samples were prepared as follows:

1. Liquor samples (containing 100 mg total extractives, see Table 3)were transferred into a 20×150 mm glass culture tubes and dried using anoven set at 75° C.2. 1 mL of cold 72% sulfuric acid was added to each tube containing 100mg of extractives/carbohydrate (bone dry basis), carefully mixed, thenincubated in refrigerator overnight (4° C.).3. Samples were heated at 30° C. for 1 hour followed by addition of 28mL of MilliQ-H₂O4. Samples were autoclaved at 121° C. for 1 hour (wet run) then cooledto room temperature.5. 20-25 mL supernatant was removed and centrifuged at 13,500 rpm for30-60 minutes at room temperature.6. Clear supernatant was removed for analysis, or stored at −20° C.High performance liquid chromatography was then performed at theAustralian Proteome Analysis Facility (APF, www.proteome.org.au)

4.2 Results Total Sugar-Acid Hydrolysis and HPLC Analysis

Tables 4-9 summarise total sugar concentration calculations andmolecular ratios of the different types of mono sugars in hemicelluloseliquor samples subjected to acid hydrolysis. Results are summarised inTable 10.

TABLE 4 Detected amount by HPAEC-PAD (pmol) Sample (i) Sample (ii)Sample (i) (control) Sample (ii) (control) Ara 192 220 112 223 Gal 32935 332 52 Glc 319 91 298 36 Xly 132 49 346 90 Man 465 21 756 29 Fru 0 870 0

TABLE 5 Concentration of sample diluted with water to 1/50 (uM) SampleSample (i) Sample Sample (ii) (i) (control) (ii) (control) Ara 19 22 1122 Gal 33 4 33 5 Glc 32 9 30 4 Xly 13 5 35 9 Man 46 2 76 3 Fru 0 9 0 0

TABLE 6 Concentration of sample (uM, x 50 dilution factor) Sample SampleSample (i) Sample (ii) MW (i) (control) (ii) (control) 150.13 Ara 9591100 561 1117 180.16 Gal 1647 176 1658 261 180.2 Glc 1593 456 1489 182150.1 Xly 661 247 1731 448 180.16 Man 2325 105 3780 147 SUM 7184 20839218 2154

TABLE 7 Molecular ratio (%) Sample Sample (i) Sample (ii) Sample (i)(control) (ii) (control) Ara 13 53 6 52 Gal 23 8 18 12 Glc 22 22 16 8Xly 9 12 19 21 Man 32 5 41 7 SUM 100 100 100 100

TABLE 8 Weight in 29 mL (mg) Sample Sample Sample (i) Sample (ii) (i)(control) (ii) (control) Ara 4.2 4.8 2.4 4.9 Gal 8.6 0.9 8.7 1.4 Glc 8.32.4 7.8 0.9 Xly 2.9 1.1 7.5 1.9 Man 12.1 0.5 19.7 0.8 SUM 36.1 9.7 46.29.9

TABLE 9 Weight ratio (%) Sample Sample Sample (i) Sample (ii) (i)(control) (ii) (control) Ara 12 49 5 49 Gal 24 9 19 14 Glc 23 25 17 10Xly 8 11 16 20 Man 34 6 43 8 SUM 100 100 100 100

TABLE 10 Overview of total sugar-acid hydrolysis Concentration (μM)Weight ratio (%) Sam- Sample Sam- Sample Sam- Sample Sam- Sample ple (i)ple (ii) ple (i) ple (ii) Sugar (i) control (ii) control (i) control(ii) control Ara 959 1100 561 1117 12 49 5 49 Gal 1647 176 1658 261 24 919 14 Glc 1593 456 1489 182 23 25 17 10 Xyl 661 247 2325 105 8 11 16 20Man 2325 105 3780 147 34 6 43 8 SUM 7184 2083 9218 2154 100 100 100 100

HPLC results demonstrated that each sample analysed was a hemicellulosefraction based on the type and ratio of mono sugars released followingacid hydrolysis. Assuming a 100 mg of total mono sugars, the percentageratios of the main sugars following acid hydrolysis for sample (ii) wereas follows: Man:Gal:Glc:Xyl:Ara=43:19:17:16:5.

Example 6 Extraction of Hemicellulose from Wood Flour Slurry andStabilisation of Lignin/Cellulose Composite to Produce a Bio-Oil Product

A stepwise process was used to extract hemicellulose from woodflour feedand produce a stable oil from the remaining lignin/cellulose composite.

Woodflour Slurry Preparation

Woodflour slurry for hemicellulose extraction was prepared fromapproximately 25 kg of woodflour. Water was added such that theresulting slurry contained approximately 18% woodflour and 82% water.

Hemicellulose Extraction

Hemicellulose was extracted from the slurry as described in Example 2above using the following conditions:

reactor temperature 190° C.,

reactor pressure 31 bar,

residence time 5 minutes,

woodflour size 150 microns (in water).

The resultant filter cake (containing lignin and cellulose) wastransferred to another reactor for further processing.

Conversion of Lignin/Cellulose to Bio-Oil Product

Filter cake containing lignin and cellulose composite derived frompre-processing was subjected to treatment with aqueous ethanol in areactor. Reaction conditions were as follows:

Reactor Temperature: 320° C. Reactor Pressure: 200 bar Woodflour SlurryRatio Estimate (by weight):  5% Additives - Ethanol (80L) 25% by volumeResidence time 18 minutes

Analysis of Bio-Oil Emulsion

The sample analyzed was water-based and contained in a PET bottle. Ittook the form of an orange-coloured emulsion. Some brown oil/tar wascoated on the wall of the bottle. A small amount of the orange emulsionwas shaken with diethyl ether, resulting in a brown ether layer and aclear, slightly coloured lower (aqueous) layer. The ether layer wasanalysed by gas chromatography mass spectrometry (GCMS) as was the brownoil/tar phase, also dissolved in ether.

Gas Chromatography Mass Spectrometry (GCMS) Results

GCMS chromatograms revealed the presence of many compounds in theemulsion. Larger peaks in the GCMS reports were integrated automaticallyand the mass spectra associated with the peaks compared with a spectrallibrary. The library compound with the closest spectral match was thenassigned to the peak by the software. Examples of compounds that werematched with a high degree of confidence include:

(i) Compounds in emulsion:

-   phenol, 2-cyclopentene-1-one,-   2-methyl, methoxyphenol,-   ethylmethoxyphenol,-   methoxypropylphenol.

(ii) Compounds in oil:

-   methoxyphenol,-   ethylmethoxyphenol,-   methoxypropenylphenol

Proton NMR Analysis

The oil sample was dissolved in d6-acetone and the proton NMR spectrumrecorded. Some of the remaining emulsion/water phase was extracted withdiethyl ether which was then removed under reduced pressure to give anorange-brown oil “ether extract”. The sample was dissolved in d6-acetoneand the proton NMR spectrum recorded.

The NMR spectra of the brown oil/tar and the ether extract were complex.The spectrum of the oil in particular had broad, ill-defined peaks. Thespectrum of the ether extract was divided approximately into 5 chemicalshift regions so that the signals could be integrated providing anapproximate idea of the types and relative abundances of functionalgroups present could be obtained (see Table 11). These abundances wererounded to the nearest whole number (except for the first row) and thepossible presence of residual solvent signals (ether, ethanol,isopropanol, acetone, water) disregarded, as were inaccuracies in theintegration.

TABLE 11 NMR spectra analysis Possible chemical Approximate Chemicalenvironment of relative abundance shift proton of protons range ArOH orRCHO 0.5   9+ Aromatic or olefin 7 9-5 Adjacent to oxygen 4 5-3Methylene or 6 3-1.2 aromatic methyl Methyl 3 <1.2A complex mixture of this nature cannot be represented by a small numberof compounds. For conceptual purposes, an equimolar mixture of the twocompounds below may produce similar integrations for the chemical shiftregions in the Table 11 above (NB: ArOH/RCHO protons were disregardedfor this purpose).The estimated chemical shifts of the protons are shown in blue

It was also observed that no wood slurry appeared to be present in theproduct emulsion inferring that all the slurry has been converted to oiland potentially gaseous products.

Example 7 Production of Bio-Oil from Radiata Pine Wood Flour (i)Reaction Conditions

To improve the quality of the bio-oil product, the effect of varyingreactor retention time and various reaction conditions was tested onradiata pine wood flour stripped of hemicellulose (see Example 2 above)in a mixture of water and 5-20% wt ethanol under pressure.

Trials were conducted under the various conditions shown in Table 12.Table 12 lists the target temperature and pressure at which the pilotplant conditions were maintained as close to as practicable.

TABLE 12 variations in reaction conditions Reactor Target Target SlurrySolids Retention Pressure Temperature Concentration (minutes) (bar) (°C.) (% wt) 15 120-240 280-350 4-30 5 120-240 280-350 4-30 30 120-240280-350 4-30

In each case, the wood flour was successfully processed to generate aliquid product containing two phases:

1. A lighter aqueous phase containing lighter dissolved organics whichcould be extraction to produce a mobile light oil.2. A heavier black oil phase.

(ii) Analyses of Product Black Oil Phase

Samples of the heavier oil produced from 15 and 30 minutes reactorretentions were analysed using gel permeation chromatography (GPC) toprovide an indication of molecular weight distributions of variouscompounds within the heavy oil. The typical measured distributions areshown in FIG. 2.

The GPC results show that increasing retention time decreases themolecular weight distribution, resulting in lighter oil.

A sample of the heavier oil produced from 15 minutes reactor retentionwas dried removing the bound water by distillation and an ultimateanalysis then performed on the dry sample. The elements tested andmeasured weight fractions are reported in Table 13. On a dry basis,radiata pine is typically 40% oxygen by weight. From the results it canbe seen that the heavy oil sample can be no more than 19% oxygen asdetermined by difference. This represents at least 50% reduction inoxygen content compared to the feedstock, greatly increasing the energyof the heavy oil compared to the starting feedstock.

TABLE 13 Ultimate Analysis of 30 minute retention heavier oil WeightElement Fraction Carbon 74.71% Hydrogen 6.39% Nitrogen 0.00% Sulphur0.00%

A sample of the heavy oil produced from 30 minutes reactor retention wasused for Thermo gravimetric analysis (TGA). Thermo gravimetric analysis(TGA) measures the mass lost from a sample during heating in a flow ofdry nitrogen. As indicated by the results in FIG. 3, the heavier oil hasa very broad boiling point range, and is quite volatile untilapproximately 400° C.

GCMS is a technique that can be used to identify compounds. A gaschromatograph (GC) is used to separate the individual compounds in asample and the output from the GC is then fed into a mass spectrometer(MS) which ionizes the compounds and measures the mass to charge ratioof the fragments. The data is then matched against a library to providea probable identification of the compounds.

FIGS. 4 and 5 show data provided by GCMS analysis of the aqueous andheavier oil phases from the 30 minute retention products. Hundreds ofcompounds are present, and the largest 10 peaks based on area have beenassigned from the NIST spectral library. The assignments provide anindication as to the nature of the oils which are largely oxygenatedaromatics. FIG. 4 shows the results of GCMS analysis of the aqueousphase. FIG. 5 shows the results of GCMS analysis of the heavier oilphase.

1-31. (canceled)
 32. A method for producing a bio-oil from lignocellulosic matter, the method comprising the steps of: (a) solvating hemicellulose from the lignocellulosic matter using a solvent; (b) removing solvated hemicellulose from solid matter remaining after step (a); and (c) solvating lignin and cellulose from the solid matter remaining after step (a) using a solvent at a reaction temperature of between 180° C. and 350° C. and a reaction pressure of between 8 MPa and 26 MPa, wherein step (c) of solvating lignin and cellulose produces the bio-oil.
 33. The method according to claim 32, wherein said lignocellulosic matter comprises 10%-35% hemicellulose, 15%-45% cellulose and 2%-35% lignin.
 34. The method according to claim 32, wherein said lignocellulosic matter comprises 20%-35% hemicellulose, 20%-45% cellulose and 20%-35% lignin.
 35. The method according to claim 32, wherein the solvent of step (c) is an aqueous alcohol comprising no more than ten carbon atoms.
 36. The method according to claim 35, wherein the aqueous alcohol is ethanol or methanol.
 37. The method according to claim 35, wherein the aqueous alcohol comprises 1%-30% alcohol by weight.
 38. The method according to claim 35, wherein the aqueous alcohol comprises about 20% alcohol by weight.
 39. The method according to claim 32, wherein step (c) is performed at a reaction temperature of between 280° C. and 350° C.
 40. The method according to claim 32, wherein step (c) is performed at a temperature of about 320° C.
 41. The method according to claim 32, wherein step (c) is performed at a reaction pressure of between 12 MPa and 24 MPa.
 42. The method according to claim 32, wherein step (c) is performed at a reaction pressure of about 20 MPa.
 43. The method according to claim 32, wherein the lignin and cellulose of step (c) is in the form of a slurry comprising between 2% and 45% solid matter by weight.
 44. The method according to claim 43, wherein the slurry comprises about 10% solid matter by weight.
 45. The method according to claim 32, wherein step (c) is performed for between 2 minutes and 60 minutes.
 46. The method according to claim 32, wherein step (c) is performed for between 5 minutes and 30 minutes.
 47. The method according to claim 32, wherein the solvating of hemicellulose in step (a) is performed at a reaction temperature of between 100° C. and 250° C., and a reaction pressure of between 0.2 MPa and 5 MPa.
 48. The method according to claim 32, wherein the solvent of step (a) is: (i) an aqueous acid and the treatment is performed at a pH of below about 6.5; (ii) an aqueous base and the treatment is performed at a pH of above about 7.5; or (iii) water.
 49. The method according to claim 32, further comprising pre-treating the lignocellulosic matter prior to solvating hemicellulose in step (a), wherein the pre-treating comprises producing a slurry comprising a mixture of a solvent and particles derived from the lignocellulosic matter.
 50. The method according to claim 49, wherein said particles are between about 100 microns and about 1000 microns in size.
 51. The method according to claim 49, wherein the slurry comprises between about 5% and about 20% lignocellulosic matter. 